Fast thermo-optical particle characterisation

ABSTRACT

The present invention relates to a method and an apparatus for a fast thermo-optical characterization of particles. In particular, the present invention relates to a method and a device to measure the stability of (bio)molecules, the interaction of molecules, in particular biomolecules, with, e.g. further (bio)molecules, particularly modified (bio)molecules, particles, beads, and/or the determination of the length/size (e.g. hydrodynamic radius) of individual (bio)molecules, particles, beads and/or the determination of length/size (e.g. hydrodynamic radius).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/515,641, which was published as U.S. 2010-0044586-A1 on 25 Feb.,2010; and entered U.S. National Stage on 20 May 2009 fromPCT/EP2007/010037, filed 20 Nov., 2007, which was published in Englishon 29 May 2008 as WO 2008/061706; and claims priority to European PatentOffice Application Nos. 06024057.9, filed 20 Nov. 2006; and 07020650.3,filed 22 Oct. 2007.

The present invention relates to a method and an apparatus for a fastthermo-optical characterisation of particles. In particular, the presentinvention relates to a method and a device to measure the stability ofmolecules, like biomolecules, the interaction of molecules, particularlybiomolecules with, e.g. further (bio)molecules, particularly modifiedbiomolecules, particles, e.g. nanoparticles or microparticles, beads,e.g. microbeads and/or the determination of the length/size (e.g.hydrodynamic radius) of individual molecules, particularly ofbiomolecules, of particles (e.g. nanoparticles, microparticles), or ofbeads (e.g. microbeads) as well as the determination of e.g. length orsize (e.g. hydrodynamic radius) of (bio)molecules or particles. Alsocombinations these characteristics may be determined with the means andmethods of this invention. It is of note that the present invention is,however, not limited the measurement/characterization of biomolecules.Therefore, also the characteristics of other compounds/particles can bemeasured and determined by the means and methods disclosed herein, forexample kinetic events and interactions of molecules may be determinedand/or measured. Accordingly, also chemical reactions (like inorganic ororganic reactions) may be measured by the methods and under use of thedevices of the present invention. It is also envisaged to determinecomplex formations and/or their dissociation.

In conventional methods known in the art for all-optical biomoleculecharacterisation, samples with biomolecules in solutions arehomogeneously heated to a certain temperature at a time followed byfurther heating to the next temperature point. A common procedure is tostart at 20° C. The temperature is then increased for example by 1° C. Awaiting time of approx. 2 minutes is then required until the wholesystem (cuvette and solution) has reached the applied temperature. Thisis due to the large thermal mass. Only then fluorescence is measured.This procedure is repeated until 90° C. in a stepwise manner.Accordingly, the heating of the whole sample volume takes long and it isnecessary to employ heat conducting materials in contact with theliquid.

Separation techniques known in the art such as gel electrophoresis areat core of modern DNA and protein biotechnology. However,electrophoresis is hard to miniaturize due to electrochemical effects atthe metal-buffer interface and the tedious preparation of the gel phase.Duhr et. al. in European Phys. J. E 15, 277, 2004 relates to“Thermophoresis of DNA determined by microfluidic fluorescence” andmakes use of thermophoretic driving forces in miniaturized biotechnologydevices. This article discusses an all-optical approach in thin microfluids to measure and apply thermophoresis for biomolecules in smallvolumes. The temperatures are measured with high spatial resolution bythe temperature sensitive fluorescence of a fluorescent dye. Typically,one measurement according to Duhr et al. (2004, loc. cit.) takes 300 sor even more. It is further speculated in Duhr et al. (2004, loc. cit.),that the movement of polymers, in particular DNA, in a temperaturegradient is independent of the chain length of the molecule, anassumption in line with theoretical considerations, see e.g. Braun andLibchaber, Physical Review Letters 89, 18 (2002). This assumptionstrongly confines thermo-optical characterization of molecules based onthermophoresis, since the technique would solely be dependent on changesin size of molecules and would exhibit no sensitivity to surfaceproperties, as it is the gist of the present invention.

The above mentioned method has the disadvantage that it is very timeconsuming. This is also the case for established methods for measuringinteractions, size and stability, like Biacore (GE Healthcare), EvotecFCS-plus (Perkin-Elmer) or Lightcycler 480 (Roche Applied Science). Thetime consumption of these techniques is typically longer than an hour.

It is therefore an object of the present invention to provide animproved method and device for a thermo-optical characterisation ofparticles or molecules, in particular to provide a very fast method tomeasure thermally-induced processes of particles or molecules, inparticular of biomolecules.

These objects are achieved by the features of the independent claims.Further preferred embodiments are characterized in the dependent claims.

The present invention relates in particular to a method and a device formeasuring the stability of molecules, in particular biomolecules, theinteraction of (bio)molecules with other or further (bio)molecules, orwith particles (e.g. nanoparticles, microparticles, beads, e.g.microbeads), and/or measuring the length or size of molecules, likebiomolecules, particles (e.g. nanoparticles or microparticles,microspheres, beads, e.g. microbeads), Also combinations of thesecharacteristics may be determined by the means and methods of thisinvention. The method of the present invention allows contact free,thermo-optical measurements of these parameters/characteristics within atime span of a few milliseconds up to a few seconds, i.e. a very fastanalysis is possible. In the context of the present invention, ananoparticle is a microscopic particle with at least one dimension lessthan 100 nm and a microparticle/microbead is a microscopic particle/beadwitch has a characteristic dimension of less than 1 mm but normally morethan 100 nm.

In the context of this invention, in particular the claims, it is notedthat the terms “particle” or “particles” also relate to beads,particularly microbeads, nanoparticles or molecules, particularlybiomolecules, e.g. nucleic acids (such as DNA, RNA, LNA, PNA), proteinsand other biopolymers as well as biological cells (e.g. bacterial oreukaryotic cells) or sub-cellular fragments, viral particles or virusesand cellular organelles and the like. The term “modified particle” or“modified bead” relates in particular to beads or particles whichcomprise or are linked to molecules, preferably biomolecules. This alsocomprises coating of such beads or particles with these (bio)molecules.

According to one embodiment of the invention, the inventive method isbased on the absorbance of infrared LASER radiation by aqueous solutionsand the subsequent conversion into heat. Thereby it is possible tocreate broad spatial, i.e., two-dimensional or three-dimensional (2D,3D), temperature distributions comprising all desired temperatures, e.g.between 0° C. and 100° C. within an area of e.g. about 250 μm indiameter or length, established by local laser heating, whereby desiredtemperature gradients are created, in particular strong temperaturegradients. Both, local temperature distributions and temperaturegradients, can be used as described below to measure parameters, inparticular biomolecular parameters. In a particular embodiment, thetemperature distribution is on the micrometer scale. This may beadvantageous since strong temperature gradients shorten theequilibration time of the system needs to equilibrate (i.e. measurementtime). In particular embodiments, it is advantageous to increase thetemperature on a length scale of less than 100 μm.

The present invention relates to a method to measure thermo-opticallythe characteristics of particles/molecules in a solution with the stepsof: providing a sample probe with marked particles/molecules in asolution; exciting (e.g. fluorescently) said marked particles andfirstly detecting and/or measuring (e.g. fluorescence of) said excitedparticles/molecules; irradiating a laser light beam into the solution toobtain a spatial temperature distribution in the solution around theirradiated laser light beam (i.e. in and/or nearby the area of thesolution which is directly irradiated by the laser light beam);detecting and/or measuring secondly (e.g. a fluorescence of) theparticles/molecules in the solution at a predetermined time afterirradiation of the LASER into the solution has been started, andcharacterizing the particles/molecules based on said two detections.

Without differing from the gist of the invention it is also envisagedthat instead of a detection based on fluorescence other detectionmethods are possible. Depending on the size and properties of theparticles to be detected, the step of fluorescently exciting may beomitted, and a detection based on light scattering, (UV) absorption,phase contrast, phosphorescence and/or polarisation are possible.Moreover, for particles larger than 100 nm, the movement of suchparticles can be detected by single particle tracking.

The thermo-optical characterization in accordance with this inventionallows to determine properties of molecules or particles in solutions,in particular in aqueous solutions. It also allows to discriminatebetween different conformations of one particle or molecule species andit also allows to discriminate between different species of particles ordifferent molecules. The characterization can be used in all cases wherethe particles show a response to changes in the temperature gradient andchanges in the absolute temperature. An advantageous feature of thepresent invention is the presence of a defined spatial temperaturedistribution. In particular, the temperature distribution is generatedlocally on microscopic length scales by local heating with a focussedlaser. Another advantageous feature is that the response of theparticles or molecules is assigned to a certain place of the known,optically generated spatial temperature distribution. Accordingly,temperature, place and response of the particles are directlycorrelated.

Furthermore, and in contrast to Duhr (2004; loc. cit.) the presentinvention provides means and methods for the thermo-optical measurementsand/or thermo-optical characterization of particles or molecules, inparticular biomolecules, by the measurements and/or the detection ofdifferences in the thermo-optical properties. Their thermo-opticalproperties mainly originate from differences in thermophoretic mobilityDT (i.e. the velocity of particles/molecules in a temperature gradient).In particular, the detected signal is dependent on the thermophoreticmobility c/c₀=exp[−(D_(T)/D)(T−T₀)], with the diffusion coefficient D,concentration c and temperature T. A DT independent of the polymerlength as expected from Duhr (2004; loc. cit.) and others (e.g. Chan etal., Journal of Solution Chemistry 32, 3 (2003); Schimpf et al.,Macromolecules 20, 1561-1563 (1987)) would render the analytics ofbiopolymers like DNA and proteins almost impossible since only changesin the diffusion constant would contribute to the thermo-opticalproperties, which are minute in most cases.

Thermo-optical characterization in accordance with this invention isbased on the creation of strong temperature gradients at microscopiclength scales in solution, in particular in aqueous solution. By doingso, the energetic states of the molecules in the solution are changeddepending on the temperature and the properties of the molecule, i.e.the molecules experience a spatial potential originating from thespatial differences in temperature. This potential drives a directedmotion of molecules along the temperature gradient, an effect calledthermophoresis. In other cases the change in temperature leads, besidethermophoresis, to an unfolding of biopolymers, like proteins or DNA.The unfolding effect is observed at high temperatures and is a measurefor the stability of molecules (the reason for unfolding is theincreased influence of the entropy component of the energy), the effectis separated from thermophoresis by a characteristic time scale. Thestability analysis takes place in milliseconds to one second, preferablyabout 1 ms to 250 ms, 1 ms to 200 ms, 1 ms to 100 ms, 1 ms to 80 ms, 1ms to 50 ms, more preferably about 40 ms, 80 ms-180 ms, 80 ms-150 ms,most preferably about 50 ms.

Thermophoresis is observed at times in a range from about 0.5 seconds to250 seconds, preferably 0.5 seconds to 50 seconds, preferably 1 secondto 250 seconds, preferably 1 second to 50 seconds, preferably 1 secondto 40 seconds, preferably 5 seconds to 20 seconds, preferably 5 secondsto 40 seconds, preferably 5 seconds to 50 seconds, preferably 5 secondsto about 80 seconds, more preferably 5 seconds to 100 seconds.Thermophoresis is a method which is sensible to surface properties ofmolecules in a solution. It is not necessary to expose molecules to adifferent matrix (like in chromatography) or to interact with themolecules physically in any way (e.g. by direct contact or by addingsubstances). Only interactions between electromagnetic waves and matterare necessary. Infrared radiation is used for spatial heating (i.e.manipulation of matter) and fluorescence to detect molecules.

The gist of thermo-optical characterization based on thermophoresis asprovided herein is that differences in thermophoretic mobility (i.e. thevelocity of molecules in a temperature gradient), and hydrodynamicradius can be detected by analyzing the spatial distribution ofconcentration (i.e. by the spatial distribution of e.g. fluorescence) orthe fluctuations of single particles trapped in the spatial temperatureprofile. This embodiment is of particular relevance for the hereindescribed thermo-optical trap for trapping particles, molecules, beads,cellular components, vesicles, liposomes, cells and the like. While thehydrodynamic radius is only related to the radius of a molecule, thethermophoretic mobility is sensitive to charge, surface properties (e.g.chemical groups on the surface), shape of a molecule (i.e. size ofsurface), conformation of a protein or interaction between biomoleculesor biomolecules and particles/nanocrystals/microbeads. This means thatif any of the mentioned properties are changed, the molecules willexperience a different thermodynamical potential, resulting indifferences in thermophoretic mobility (i.e. change in spatialconcentration profile or fluctuation amplitude of trapped particles).

Thus, the present invention relates to thermally induced processes, e.g.temperature gradient induced directed motion or thermal denaturation.

The thermo-optical characterization mentioned above provides the meansfor fast thermo-optical analysis of particles and/or molecules, inparticular for the thermo-optical characterisation of biomolecules, likenucleic acid molecules (e.g. DNA, RNA, PNA) or proteins and peptides.This characterisation comprises, inter alia, size determination, lengthdetermination, determination of biophysical characteristics, likemelting points or melting curves, complex formations, protein-proteininteractions, protein or peptide folding/unfolding, of intra-molecularinteractions, intermolecular interactions, the determination ofinteractions between particles or molecules, and the like. Prior artmethods for detection and quantification of molecular interactions andcharacteristics, in particular biomolecular interactions andcharacteristics are very time consuming which means that the time neededfor an analysis is in the order of 30 minutes up to hours. In accordancewith the present invention, one measurement typically takes less than300 s, less than 200 s, less than 100 s or even less than 50 s, which isclearly faster than the methods described in the prior art. The presentinvention can detect and quantify molecular interactions andcharacterisations, in particular biomolecular interactions and/orbiochemical/biophysical properties within 1 second to 50 seconds. Theterm interaction comprises interaction between biomolecules (e.g.protein, DNA, RNA, hyalauronic acids etc.) but also between (modified)(nano)particles/(micro)beads and biomolecules. In this context, modifiedparticles, molecules, biomolecules, nanoparticles, microparticles, beadsor microbeads comprise fluorescently labelled particles, molecules,biomolecules, nanoparticles, microparticles, beads or microbeads.Fluorescently labelled particles, molecules, biomolecules,nanoparticles, microparticles, beads or microbeads may be e.g.particles, molecules, biomolecules, nanoparticles, microparticles, beadsor microbeads, to which one or more fluorescent dyes have been attached,e.g. covalently attached. For example, the fluorescent dyes may beselected from the group of6-carboxy-2′,4,4′,5′,7,7-hexachlorofluorescein (6-HEX SE; C20091,Invitrogen), 6-JOE SE or 6-TET SE (see also appended FIG. 6). In othercases, the intrinsic fluorescence of e.g. particles, molecules,biomolecules may be exploited according to the invention, e.g. thefluorescence properties of tryptophan, tyrosine or phenylalanine in aprotein may be exploited. The terms “marked” and “labelled” are usedsynonymously in the context of this invention. In the context of thisinvention, “marked particles” refer to fluorescently labelledmolecules/particles or other molecules/particles which can be detectedby fluorescence means, e.g. molecules/particles comprising an intrinsicfluorophor, molecules/particles comprising intercalating dyes orparticles/molecules with fluorophores attached.

A typical experiment in accordance with this invention, but not limitingthe scope of the invention, to detect/quantify interactions may bedescribed as follows:

Step 1a, Background Measurement:

A sample buffer without fluorescently labelled samplemolecules/particles is filled into a microfluidic chamber and thefluorescence is measured, while the excitation light source is turnedon.

Step 1b, Determination of Fluorescence Level Before Laser Heating:

An aqueous solution of a fluorescently labelled sample (e.g.biomolecules, particles, like nanoparticles or microparticles, beads,particularly microbeads, wherein in particular embodiments all of themhave a specific affinity for other biomolecules) at a givenconcentration is filled in the microfluidic chamber (preferably acapillary) which preferably provides a defined height of the chamber.Fluorescence is excited and recorded with (e.g. CCD-Camera) or without(e.g. Photomultiplier tube, Avalanche Photodiode) spatial resolution forless than 10 seconds e.g. on a CCD device or photomultiplier withexposure times of 25 milliseconds up to 0.5 seconds. Then thefluorescence excitation is turned off.

Step 2, Starting of Infrared Laser Heating:

The infrared heating laser is turned on and the spatial temperaturedistribution is established within a few milliseconds within thesolution. The temperature gradient has been calibrated once and it isnot necessary to repeat this calibration every time an experiment isperformed. In particular a setup where infrared heating and fluorescenceimaging are performed through the same optical element from one side isadvantageous for the stability of the optical and infrared foci.

A decrease of fluorescence due to photobleaching lower than 5% isadvantageous in the experiment. In particular embodiments of theinvention, no correction for photobleaching is necessary.

In some particular embodiments for measuring thermophoretic properties,the maximal temperature is below the temperature which is known to causedamage to the molecules in the solution or disturb their interaction(e.g. temperatures between 1 and 5° C. above ambient temperature).

Depending on the thermophoretic properties of the particles or moleculesin the solution (i.e. if they move fast in a thermal gradient or slow)the infrared laser heats the solution for 5 seconds up to 100 seconds,preferably for 5 seconds up to 50 seconds, more preferably for 5 secondsup to 20 seconds.

Step 3, Recording of the Spatial Fluorescence (i.e. Concentration)Profile:

After this period of time, the fluorescence excitation is turned on andimages are recorded with the same frame rate and length as described instep 1b. Step 3 is the last acquisition step necessary for evaluation ofthermo-optical properties.

For detection and quantification of interactions more measurementsfollowing the protocol described previously are necessary. Step 1a isrepeated with sample buffer and in step 1b the aqueous solution of afluorescently labelled sample is mixed with an amount of the biomoleculewith which the interaction should be detected or quantified. Forexample, in the detection of an interaction of particles and/ormolecules, the fluorescently labelled sample (comprising one bindingpartner) is mixed with a sufficient amount of the second binding partnerso that a substantial amount of the fluorescently labelled molecule orparticle is in the complex with the binding partner. If the strength ofthe interaction is to be quantified in terms of e.g. a dissociation orassociation constant (Ka, Kd), than the procedure described previouslymay be conducted with varying concentrations of binding partner (e.g.0.1×-10× the concentration of the fluorescently labelled bindingpartner). This means that a titration of binding partner may beperformed.

Processing the Raw Data:

Optionally, a (linear) bleaching correction can be performed for whichit is advantageous to wait for the back-diffusion of all moleculesfollowing the end of step 3. This increases the time consumption of theanalysis dramatically. For precise and fast measurements it isadvantageous to determine the strength of bleaching from image to imageand correct every individual image with its own bleaching factor. For aprecise bleaching correction it is advantageous that the temperaturegradient at distance from the heat spot is low (e.g. below 0.001 K/μm).The images taken in step 1b are used to correct all images forinhomogeneous illumination. In case fluorescence is recorded withoutspatial resolution (e.g. avalanche photodiode or photomultiplier)photobleaching is corrected best by determining once the bleachingcharacteristic of a certain dye without heating laser in a controlexperiment.

Data Evaluation:

Qualitative detection of interaction: From the image series the spatialfluorescence distribution of the reference experiment (i.e.fluorescently labelled molecule/particle without binding partner) andthe second experiment (i.e. were the binding partner is present) isextracted. The fluorescence is plotted versus the distance from the heatspot. An averaging is only possible for pixels with the same temperatureand same distance. The spatial concentration distribution is obtained bycorrecting the fluorescence intensities for the respective temperaturedependence of the dye. With knowledge of temperature dependence of thefluorescent dye and the spatial temperature distribution, the effect ofa decreasing fluorescence due to temperature increase can be corrected.In particular embodiments, a correction for temperature dependency isnot necessary for the qualitative detection of interaction as well astheir quantification, and the spatial fluorescence distribution issufficient. Any fluorescent dye on the market may be used, in particularembodiments even without characterization of its temperature dependency.The fluorescent properties of a dye may vary with buffer conditions,such as pH.

The values of the fluorescence profile are integrated up to the distancewere the temperature is below e.g. 10% of the maximum temperature (e.g.70 μm). The integrated values are compared and a change give a preciseindication if there is an affinity between the substances at theconcentrations used, since the interaction changes the thermo-opticproperties (e.g. thermophoretic mobility, surface size and chemicalgroups on surface). In most cases, the interaction leads to higherfluorescence (concentration) at higher temperatures

In case the whole cross-section of a capillary is heated (i.e. usinge.g. cylindrical lenses to give the IR laser beam a ellipsoidal shape,which heats a cross section of a capillary homogeneously), the intensityof two or more pixels from the centred heat spot may be averaged. Inparticular embodiments all pixels at the same distance to the heatedline have the same temperature. This is advantageous for high precisionmeasurements. In case fluorescence is recorded without spatialresolution, the fluorescence change in the centre of the heat spot/lineis measured. In particular embodiments it may be advantageous to heatthe whole cross section. In general if more than a single frame isrecorded in step 1b and 3 an integration of multiple frames is possible.

For a quantification of molecular affinities or particle affinities thesame procedure is performed for all experiments at variousconcentrations of non-fluorescent binding partner. The result of theintegration for the reference experiment (i.e. without binding partner)is subtracted from the integrated values obtained for the differentconcentrations of binding partners. From this evaluation, the amount ofinteracting complexes in arbitrary units may be obtained. By dividingthese values by the value where binding is saturated, the relativeamount of formed complexes between the interacting molecules,particularly the binding partner, at a certain concentration of bindermay be obtained. From these datasets also the concentration of free,e.g. non-fluorescent, binding partner may be determined and the strengthof the interaction may be quantified in terms of association ordissociation constant (see also appended examples).

As mentioned previously, it is also possible to detect the binding ofmolecules to larger inorganic particles or nanocrystals using theprocedure described previously and herein below. Inorganic particles,e.g. CdSe particles, may be modified with a varying numbers (e.g. 1, 2,3, or 3 or more, yet preferably up to 3) of Poly-ethylen-glycol (PEG) ofdifferent molecular weight. In particular embodiments, 1 to 3Poly-ethylen-glycol (PEG) molecules are attached to the particles. Thespatial fluorescence profile is measured as described herein for thedetection of biomolecular interactions (see appended examples). Also theraw data are processed as described herein. To measure the number orsize of PEG molecules bound to the particles or nanocrystals, it may besufficient to compare the spatial fluorescence profiles obtained withthe protocol described previously. However, a correction for thetemperature dependent decrease of the fluorescence allows aquantification in terms of the Soret coefficient. It is illustrated inthe appended figures and examples, particularly in appended FIG. 26,that the Soret coefficient increases linearly with the number of PEGmolecules bound to the nanocrystals. The slope of the increase dependson the molecular weight of the PEG. The binding of single molecules ofthe size of a protein is detectable as illustrated in e.g. appended FIG.26.

The terms “interaction” or “affinity” as used herein and in particularin the above outlined, non-limiting illustrative experiment hot onlyrelates to the interaction of distinct molecules/particles (e.g.intermolecular interactions), but also to intramolecular interactions,like protein folding events and the like.

It is understood by the person skilled in the art that the term“fluorescence” as employed herein is not limited to “fluorescence” perse but that the herein disclosed means, methods and devices may also beused and employed by usage of other means, in particular, luminescence,like phosphorescence. Accordingly, the term “exciting fluorescently saidmarked particles and firstly detecting and/or measuring fluorescence ofsaid excited particles” relates to the “excitation step” in the aboveidentified method and may comprise the corresponding excitation ofluminescence, i.e. excitation is carried out with a shorter wave lengththan detection of the following emission. Therefore, the term “detectingand/or measuring secondly a fluorescence of the particles” in context ofthis invention means a step of detection said emission after excitation.The person skilled in the art is aware in context of this invention thatthe “excitation”-wave length and the “emission”-wave length have to beseparated.

According to a first illustrative embodiment of the present invention,the predetermined time (after which (e.g. a fluorescence of) theparticles/molecules in the solution are detected and/or measuredsecondly) is small enough that concentration changes induced bythermophoresis and artefact related to or due to convection arenegligible small. In other words, the predetermined time is short enoughto separate the inter- and intra-molecular reaction time scale fromslower temperature effects, e.g. thermophoresis, thermal convection.Thus, the predetermined time is preferably within the range of from 1 msto 250 ms, more preferably between 80 ms and 180 ms, in particular 150ms. In particular, in cases where the solution is provided in a chamberwith good thermal conductivity, e.g. sapphire, diamond, and/or silicon,a shorter predetermined time is already sufficient, e.g. 1 ms, 5 ms, 10ms or 15 ms. For the measurement it is advantageous that the chamber andthe solution are in a thermal equilibrium. In other words, for chamberswith a good thermal conductivity, the solution and the chamber arefaster in a thermal equilibrium such that shorter predetermined timesare sufficient. In case the thermal conductivity is poor, it takeslonger until the chamber and the solution are in thermal equilibrium,i.e. the predetermined time is longer, e.g. 100 ms to 250 ms.

In particular embodiments of the invention, the detection or exposuretime is in the range of from 1 ms to 50 ms. The time in which thedetection signal is recorded must be short enough that the change ofposition of an individual molecule is negligible for the detectionduring the detection step. For example, in case the detection isconducted with a CCD camera with a resolution of 320×200 pixel, it isadvantageous that during detection time an individual molecule/particlewill be detected by only one pixel, since each pixel represents acertain temperature. If the position of a particle changes too much,i.e. more than one pixel, the particle may be exposed to a differenttemperature which decreases the measurement accuracy. The use of a CCDcamera device for detection also comprises the use of a camera with onlya single line of pixel (e.g. line camera) for one-dimensional detection.

In a particular embodiment of the invention, the laser beam is defocusedsuch that a temperature gradient within the temperature distribution isin the range of from 0.0 to 2 K/μm, preferably from 0.0 to 5 K/μm. Thus,small temperature gradients ensure that the thermophoretical particlemovement is negligible small during the time from the start of laserirradiation to the end of detection.

At least all temperatures needed to detect the thermal denaturation of amolecule have to be within the field of view of the camera device.

According to a further aspect of the present invention, the laser beamis irradiated through one or a plurality of optical elements into thesolution. The focusing of the laser beam is in some embodimentsperformed in such a way that the temperature gradients lie within theabove defined ranges. Focusing of the laser can be achieved e.g. by asingle lens, a plurality of lenses or a combination of a optical fibreand a lens or a plurality of lenses or an objective where the divergenceof the incident laser beam is adjusted properly. Moreover, furtheroptical elements for controlling the focus and or direction of the laserbeam may be arranged between the solution and the laser.

According to yet a further embodiment of the present invention, thetemperature distribution around the laser beam is measured by anadditional measurement, e.g. the temperature distribution is measuredunder the same conditions on the basis of the knowntemperature-dependent fluorescence of a dye, as illustrated in theappended figures; in particular FIGS. 3 a, 3 b and 15). In particular, atemperature distribution may be determined based on detectedfluorescence of the temperature sensitive dye, wherein said temperaturesensitive dye is heated (via the solution) by the irradiated laser beamand the fluorescence spatial fluorescence intensity is measuredsubstantially perpendicular around the laser beam.

According to a second illustrative embodiment of the present invention,the predetermined time (after which (e.g. a fluorescence of) theparticles/molecules in the solution are detected and/or measuredsecondly) is sufficiently long so that changes of concentration based onthe thermophoretical motion can be detected. Thus, the predeterminedtime is preferably within the range of from 0.5 to 250 s. In saidpredetermined time the concentration changes within the spatialtemperature distribution in the solution due to thermophoretic effectsand such an concentration change may be detected by a change of thedistribution of fluorescence.

In particular embodiments of the invention the laser beam is focusedsuch that a temperature gradient within the temperature distribution isachieved in the range of from 0.001 to 10 K/μm. The temperature withinthe field of view (particularly at the edge of the field of view) doesnot necessarily reach the value of ambient temperature. A temperatureincrease at distance to the heat centre (i.e. at the edge of the fieldof view) of 10% or less (in ° C.) in comparison to the maximaltemperature (in ° C.) is advantageous.

According to a further embodiment, the fluorescence before and afterirradiation the laser is detected with a CCD camera. The use of a CCDcamera provides the advantage that the concentration change can bedetected at a plurality of positions simultaneously. In particularembodiments, the CCD camera is a 2D (two dimensional) CCD camera, i.e.,the CCD array comprises a plurality of sensor pixels (photoelectriclight sensors) in a first and a second direction, wherein the first andsecond directions are preferably perpendicular to each other. Accordingto a further embodiment, the CCD camera is a line or line-scan camera,i.e., the CCD array comprises a plurality of sensor pixels in a firstdirection (a line of sensor pixels) but merely one pixel in a seconddirection. Such a camera is also referred to as 1D (one dimensional) CCDcamera. In other words, a one-dimensional array, used in line-scancameras, captures a single slice or line of an image, while atwo-dimensional array, captures a whole 2D image. The CCD array of theline camera may also comprise three sensor lines, each for one colourchannel (Red, Green, and Blue). However, according to a further aspectof the present invention, it is also possible to measure thecharacteristics of the particles based on a detected fluorescence changeof a single pixel of the CCD. Thus, it is also possible to use aphotodiode or a photomultiplier instead of a single pixel of the CCD. Insome embodiments the brightness of the fluorescence before and afterirradiation the laser is measured with a photodiode or a single pixelwith the CCD in the centre of the laser beam.

By imaging on a CCD camera device, a line camera or a PMT/AvalanchePhotodiode, the fluorescence may be averaged throughout the height ofthe used liquid sheet. Accordingly, the three-dimensional solution maybe reduced to two dimensions. Therefore, the method described in theembodiments is also applicable to two-dimensional lipid sheets,typically used as models system for membrane processes (e.g. surfacesupported tethered bilayer lipid membrane (tBLM, as illustrated in theappended figures, particularly FIG. 38), or classical Langmuirmonolayers). Fluorescently labelled compounds floating in this membrane(e.g. lipids, proteins and alike) move in temperature gradients andrearrange according to their solvation energy. The fluorescenceredistribution in these lipid layers or membranes may be employed in thecontext of the invention, like the redistribution of compounds insolution to detect biochemical or biophysical properties orcharacteristics, like conformational changes, interactions, hydrodynamicradius and the like. In the membrane system, the local temperaturedistribution is established by the surrounding aqueous solution, e.g.the aqueous solution above a surface supported membrane. By heatconduction the lipid layer above a aqueous solution also adopts thecorresponding temperature.

According to the first and second illustrative embodiments of theinvention, the particles can be biomolecules and/or (nano- ormicro-)particles and/or beads, particularly microbeads, and combinationsof those. With the use of modifiednanoparticles/microparticles/microbeads proteins, DNA and/or RNA can bedetected by specific bindings of proteins, DNA and/or RNA to thenanoparticles/microbeads, since the specific binding changes thethermophoretic motion of the nanoparticles/microbeads. The velocity ofparticles larger than 100 nm can be detected by single particletracking.

The laser light may be within the range of from 1200 nm to 2000 nm. Thisrange is advantageous for aqueous solutions. The hydroxyl group of waterabsorbs strongly in said wavelength range. Also other solvents with ahydroxyl group like glycerol can be heated by infrared laser heating).The laser is in some embodiments a high power laser within the range offrom 0.1 W to 10 W, preferably from 1 W to 10 W, more preferably from 4W to 6 W. In some embodiments the particle concentrations of an aqueoussolution are within the range of from 1 atto Molar (e.g. single particlemicrobeads) to 1 Molar, preferably from 1 atto Molar to 100 μMolar.

According to a further embodiment, the solution may be a saline solutionwith concentrations in the range of from 0 to 1 M.

According to still a further embodiment of the invention, the spatialtemperature distribution generated by the LASER beam is in a range of0.1° C. to 100° C. The temperature sensitivity of the material ofinterest sets the limits to the maximum temperature used in experiments.In particular temperature ranges of 0.1° C. to at least 40° C.,preferably to at least 60° C., more preferably to at least 80° C. andeven more preferably up to at least 100° C. are generated by the LASERbeam to measure the DNA stability for example. The person skilled in theart is aware that corresponding temperatures can be achieved by, e.g.,cooling of the experimental system as well as by use of LASER withcorresponding power. The person skilled in the art is also aware that bycooling the overall sample a higher amplitude of temperature increase(i.e. by laser heating) is possible without causing damage totemperature sensitive materials. Illustrative, but not limiting,temperature ranges and maximum temperatures for different materials andthermo-optical characterizations is given in table 1. Accordingly, alsohigher temperatures can be achieved in the centre of the heat gradient(point of maximum laser power in and on the sample to beanalyzed/characterized) as, inter alia, illustrated in appended FIG. 3.Such high temperatures may be achieved in high pressure chambers.Moreover, in some embodiments a temperature gradient is created withinthe range of from 0.1 μm to 500 μm in diameter around the LASER beam.

TABLE 1 Illustrative temperature ranges for thermo-optical analyticsthermo-optic char.* conformation, Interaction, Hydrodynamic- structure,chemical Sample Material Stability analysis kinetic events Radius,length modification Thermo-optic Trap nucleic acids p. 0° C.-100° C. p.0° C.-80° C. p. 0° C.-80° C. p. 0° C.-100° C. p. 0° C.-80° C. (DNA, RNAetc.) mr. p. 20° C.-95° C. mr. p. 20° C.-80° C. mr. p. 20° C.-80° C. mr.p. 10° C.-90° C. mr. p. 20° C.-60° C. mr. p. 30° C.-80° C. mr. p. 20°C.-40° C. mr. p. 20° C.-40° C. ms. p. 20° C.-90° C. mr. p. 20° C.-40° C.ms. p. 30° C.-85° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C. ms. p.20° C.-30° C. proteins p. 0° C.-100° C. p. 0° C.-40° C. p. 0° C.-60° C.p. 0° C.-80° C. p. 0° C.-40° C. mr. p. 20° C.-95° C. mr. p. 0° C.-30° C.mr. p. 0° C.-40° C. mr. p. 20° C.-60° C. mr. p. 10° C.-30° C. ms. p. 30°C.-85° C. mr. p. 10° C.-20° C. mr. p. 10° C.-40° C. mr. p. 20° C.-40° C.mr. p. 15° C.-30° C. mr. p. 15° C.-20° C. ms. p. 20° C.-30° C. ms. p. 0°C.-60° C. ms. p. 20° C.-30° C. ms. p. 20° C.-25° C. vesicles, p. 0°C.-100° C. p. 0° C.-40° C. p. 0° C.-60° C. p. 0° C.-80° C. p. 0° C.-40°C. liposomes mr. p. 20° C.-95° C. mr. p. 10° C.-40° C. mr. p. 0° C.-40°C. mr. p. 20° C.-60° C. mr. p. 10° C.-30° C. ms. p. 30° C.-85° C. mr. p.20° C.-40° C. mr. p. 10° C.-40° C. mr. p. 20° C.-40° C. mr. p. 15°C.-30° C. ms. p. 30° C.-40° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C.ms. p. 20° C.-30° C. ms. p. 20° C.-25° C. (Micro-)particles p. 0°C.-100° C. p. 0° C.-60° C. p. 0° C.-80° C. p. 0° C.-100° C. p. 0° C.-80°C. (e.g. silica, mr. p. 20° C.-95° C. mr. p. 10° C.-50° C. mr. p. 0°C.-60° C. mr. p. 10° C.-60° C. mr. p. 20° C.-60° C. polystyrene, etc)ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p. 10° C.-50° C. mr. p.20° C.-40° C. mr. p. 20° C.-40° C. ms. p. 20° C.-30° C. ms. p. 20°C.-30° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C. PEG p. 0° C.-100° C.p. 0° C.-100° C. p. 0° C.-80° C. p. 0° C.-100° C. p. 0° C.-80° C. mr. p.20° C.-95° C. mr. p. 10° C.-50° C. mr. p. 0° C.-60° C. mr. p. 10° C.-60°C. mr. p. 20° C.-60° C. ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p.10° C.-50° C. mr. p. 20° C.-40° C. mr. p. 20° C.-40° C. ms. p. 20°C.-30° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C.Sugar Polymers p. 0° C.-100° C. p. 0° C.-100° C. p. 0° C.-60° C. p. 0°C.-100° C. p. 0° C.-80° C. (e.g. alginate mr. p. 20° C.-95° C. mr. p.10° C.-50° C. mr. p. 0° C.-50° C. mr. p. 10° C.-60° C. mr. p. 20° C.-60°C. hyaluroic acids) ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p. 10°C.-40° C. mr. p. 20° C.-40° C. mr. p. 20° C.-40° C. ms. p. 20° C.-30° C.ms. p. 20° C.-40° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C.two-dimensional p. 0° C.-100° C. p. 0° C.-40° C. p. 0° C.-60° C. p. 0°C.-80° C. p. 0° C.-40° C. lipidsheets (e.g. mr. p. 20° C.-95° C. mr. p.10° C.-40° C. mr. p. 0° C.-40° C. mr. p. 20° C.-60° C. mr. p. 10° C.-30°C. containing ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p. 10°C.-40° C. mr. p. 20° C.-40° C. mr. p. 15° C.-30° C. proteins) mr. p. 30°C.-40° C. ms. p. 20° C.-30° C. ms. p. 0° C.-60° C. ms. p. 20° C.-30° C.ms. p. 20° C.-25° C. (Nano-)particles p. 0° C.-100° C. p. 0° C.-100° C.p. 0° C.-80° C. p. 0° C.-100° C. p. 0° C.-80° C. mr. p. 20° C.-95° C.mr. p. 10° C.-50° C. mr. p. 0° C.-60° C. mr. p. 10° C.-60° C. mr. p. 20°C.-60° C. ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p. 10° C.-50° C.mr. p. 20° C.-40° C. mr. p. 20° C.-40° C. ms. p. 20° C.-30° C. ms. p.20° C.-30° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C. Inorganic Carbonp. 0° C.-100° C. p. 0° C.-100° C. p. 0° C.-80° C. p. 0° C.-100° C. p. 0°C.-80° C. compounds (e.g. mr. p. 20° C.-95° C. mr. p. 10° C.-50° C. mr.p. 0° C.-60° C. mr. p. 10° C.-60° C. mr. p. 20° C.-60° C.carbon-nanotubes, ms. p. 30° C.-85° C. mr. p. 20° C.-40° C. mr. p. 10°C.-50° C. mr. p. 20° C.-40° C. mr. p. 20° C.-40° C. Buckyballs etc.) ms.p. 20° C.-30° C. ms. p. 20° C.-30° C. ms. p. 20° C.-30° C. ms. p. 20°C.-30° C. *The temperatures denoted here give the preferred temperatureranges of the sample at which thermo-optical properties may be measured.The temperature increase induced by laser heating may comprise only asmall portion of the overall temperature, e.g. a thermo-opticalcharacterization of nanoparticles can be carried out at 75° C. overallsample temperature with an increase in temperature by laser heating of5° C.; a protein characterization may be carried out in a sample cooledto 10° C. and heated with an infrared laser to a maximum temperature of30° C. p.: preferably, mr. p.: more preferably, ms. p.: most preferablyIn context of the invention, the term LASER is equivalent to the term“laser” and vice versa.

The present invention also relates to a device for measuringthermo-optically characteristics of particles in a solution. Such adevice comprises a receiving means for receiving particles/molecules,particularly marked or labelled particles/molecules, within thesolution, means for exciting the particles/molecules, particularly themarked or labelled particles/molecules, means for detecting theexcitation of the particles/molecules, particularly the marked orlabelled particles/molecules, and means for obtaining a spatialtemperature distribution in the solution. A further device also referredto as device according to the present invention for measuringthermo-optically the characteristics of particles/molecules in asolution comprises a receiving means for receiving marked or labelledparticles/molecules within a solution, means for fluorescently excitingthe marked or labelled particles/molecules, means for detecting theexcited fluorescence in said solution, and a laser for irradiating alaser light beam into the solution to obtain a spatial temperaturedistribution in the solution around the irradiated laser light beam.Particularly, the laser light may be focussed locally into the solutionand the spatial temperature distribution may be obtained by theconduction of the absorbed energy as heat in the solution. By adjustingthe focus width of the electromagnetic IR radiation, the spatialdimensions of the temperature distribution can be adjusted, i.e. a broador a narrow temperature distribution is achieved. Further adjustments tothe geometry of the temperature distribution can be achieved by choosinga material for the microfluidic chamber/capillary which exhibits acertain heat conductivity (e.g. high heat conductivity, narrowtemperature distribution and vice versa). According toc/c₀=exp[−(D_(T)/D)(T−T₀)] the steady state amplitude of thethermo-optical signal is exponentially related to the increase intemperature. Using the relation shown above, a spatial temperaturedistribution can be fitted precisely to the spatial concentrationdistribution by adjusting the D_(T) and D coefficients. The time ittakes the system to reach the steady state has, beside a dependence onD, also a strong dependency on D_(T) and the temperature gradient. Theproduct of temperature gradient and the thermophoretic mobility D_(T)gives the velocity with which particles move along the temperaturegradient. As a rule-of-thumb, the stronger the temperature gradient andthe higher the thermophoretic mobility the shorter is the time needed tomeasure the thermo-optical properties. Therefore, it is advantageousthat a temperature distribution is established on microscopic lengthscales (e.g. 250 μm) to obtain a strong temperature gradient.

According to further embodiments of the present invention, the means forexciting, preferably fluorescently exciting the particles/molecules ormarked particles/molecules may be any suitable device selected from thegroup consisting of laser, fibre Laser, diode-laser, LED, Halogen,LED-Array, HBO (HBO lamps are, e.g., short arc lamps in which thedischarge arc fires in an atmosphere of mercury vapour under highpressure), HXP (HXP lamps are, e.g., short arc lamps in which thedischarge arc burns in an atmosphere of mercury vapour at very highpressure. E.g., in contrast to HBO lamps they are operated at asubstantially higher pressure and they employ halogen cycle. HXP lampsgenerate UV and visible light, including significant portion of redlight). Further means for exciting as referred to in the specificationare also preferably used.

According to particular embodiments of the present invention, the meansfor detecting the excited particles, particularly for detecting thefluorescence, in the solution may be any suitable device selected fromthe group consisting of CCD camera (2D or line-scan CCD), Line-Camera,Photomultiplier Tube (PMT), Avalanche Photodiode (APD), CMOS-Camera.Further means for detecting as referred to in the specification are alsoused in particular embodiments.

The receiving means for receiving the particles, particularly the markedparticles within a solution may be a chamber, a thin microfluidicchamber, a cuvette or a device for providing the sample in form of asingle droplet. It is advantageous to provide the sample probe within achamber which has a thickness in direction of the laser light beam from1 μm to 500 μm, in particular 1 μm to 250 μm, in particular 1 μm to 100μm, in particular 3 μm to 50 μm, in particular 5 μm to 30 μm. A personskilled in the art will understand that the term chamber also relates toe.g. a capillary, microfluidic chip or multi-well plate. In someembodiments the chamber has the same width as the dimension in directionof the laser light (e.g. a capillary). In combination with acorresponding ellipsoidal laser heating geometry such a system can bereduced from radial symmetry to a single dimension since a CCD camerawith only a single line of pixels (line-scan CCD) can be used tointegrate the fluorescence of the whole width of the chamber. In aparticular embodiment, receiving means for receiving the markedparticles within a solution alias specimen holder is attached to anoptical element, such as an objective. Such a setup avoids relativemovements of the specimen holder with respect to the objective. Furthermeans for receiving as referred to in the specification are also used insome embodiments.

The laser for irradiation the laser light may e.g. be an IR laser, e.g.,a laser with a wavelength between 1200 and 2000 nm, preferably of 1455nm and/or 1480 nm and a radiation power of 0.1 to 10 w. The light of theLaser may be coupled into the device of the present invention by meansof optical units like laser fibres (single mode or multimode) with orwithout collimator. Further means for irradiating as referred to in thespecification are also used in some embodiments and are within thenormal skill of the artisan.

The device according to the present invention may further comprise acontrol unit for controlling the means for exciting the particles and/orthe means for detecting the excited particles. In particular, thecontrol unit is adapted to enable the device of the present invention toperform the method steps as discussed with regard to the presentinventive method.

The control unit may control the type (e.g. wavelength), the intensity,the duration, and/or the time for start and stop of the irradiation ofthe means for the excitation. For instance, in a particular embodimentin which the means for excitation is a laser, the duration and/or thestart and stop time for generating a laser beam may be controlled by thecontrol unit.

The control unit may further or alternatively control the exposition,the sensitivity, the duration, and/or the time for start and stop timefor detecting/measuring by means of the means for detecting. Forinstance, in a particular embodiment in which the means for detecting isa CCD camera, the exposition timing of the CCD camera may be controlledby the control unit.

The control unit may be further adapted to control the means fordetecting dependent on functional state of the means for exciting. Inparticular, it may be advantageous to synchronize the timing ofexcitation with the timing of the detection. For instance, in aparticular embodiment in which the mean for excitation is a laser andthe means for detecting is a CCD camera, the exposure timing of the CCDis synchronized with the irradiation of the laser. This may be achievedby controlling the CCD and the laser directly, e.g. by switching on andoff the CCD and the laser synchronously.

The control unit may alternatively or additionally control means, inparticular optical means, which are arranged between the means for theexcitation and the receiving means and/or are arranged between the meansfor the detecting and the receiving means.

The device according to the present may comprise at least a shutterarranged between the receiving means for receiving the markedparticles/molecules and the means for exciting the particles/molecules,in particular the laser. The device according to the present mayadditionally or alternatively comprise at least a shutter arrangedbetween the receiving means for receiving the marked particles and themeans for detecting the particles/molecules, in particular the CCDcamera. Such a shutter may be controlled by the control unit in order toadapt the timing of the step of excitation with the timing of thedetection.

A single control unit may fulfil the functions of several items, i.e.the control unit may comprise a plurality of subunits which are adaptedfor controlling particular means.

The device according to the present may further comprise at least a beamsplitter and/or a mirror, e.g. a dichroic filter or dichroic mirror,i.e. a colour filter used to selectively pass light of a small range ofcolours while reflecting other colours, or a AOFT. The dichroic mirrormay reflect short wavelength (reflectance >80%) and transmit longwavelength (transmission >80%). The dichroic mirror may e.g. comprise anIR-transmission larger than 90% and a at least a reflectance forwavelength between 350 to 650 nm. In some embodiments, instead of adichroic mirror a silver mirror may be used. The (dichroic) mirror maybe arranged in a fixed position within the device. However, according tosome embodiments, the (dichroic) mirror may be movable, e.g. driven by adriving means (which may be controlled by the control means).

The device according to the present may comprise at least an emissionand/or excitation filter (band pass/long pass) for filtering specificwavelength.

In other words, the device according to the present invention may alsocomprise optical means arranged between the receiving means forreceiving the marked particles and the means for detecting the particlesand/or between the receiving means for receiving the marked particlesand the means for exciting the particles. Such optical means may beadapted for controlling the propagation direction of light by way oftransmission or reflection and/or for filtering or separating differentwave length by (dichroic) filters. Such optical means may be passiveoptical means or active optical means which may be controlled by thecontrol unit. For instance, there may be arranged a scanning module(e.g. a Galvano scanning mirror) between the receiving means forreceiving the marked particles and the means for exciting the particles.The scanning range and timing of such a scanning module may becontrolled by the control unit, preferably in dependency with thecontrol of the means for receiving and/or means for exciting.

Optical means which are advantageous for the device according to thepresent invention, in particular to perform the method steps of thepresent invention are exemplified above and below. In particular aplurality of optical means which are useful for a device of the presentinvention are illustrated in the detailed description of the invention.

According to a further embodiment of the invention, the irradiation ofthe laser and the detection of the fluorescence is conducted fromdifferent directions, e.g. the irradiation is from below and thedetection is from above the sample (as illustrated in the appendedfigures, particularly FIG. 1). However, the means for irradiation andthe detection can be arranged on the same side with respect to thesample probe (see e.g. FIG. 2). The device according to the presentinvention may have any orientation with respect to the direction ofgravity, i.e., the device may e.g. be oriented substantiallyperpendicular, parallel or anti-parallel with respect to the directionof gravity.

In particular embodiments of the invention the sample probe is providedin a chamber. The thickness of the sample probe in the chamber indirection of the laser light beam is preferably small, e.g. from 1 μm to500 μm, in particular 1 μm to 250 μm, in particular 1 μm to 100 μm, inparticular 3 μm to 50 μm, in particular 5 μm to 30 μm. A person skilledin the art will understand that the term chamber also relates to e.g. acapillary, microfluidic chip or multi-well plate. In a furtherembodiment the chamber has the same width as the dimension in directionof the laser light (e.g. a capillary). In combination with thecorresponding ellipsoidal laser heating geometry such a system can bereduced from radial symmetry to a single dimension. A CCD camera withonly a single line of pixels can be used to integrate the fluorescenceof the whole width of the chamber. Furthermore only a single pixel (or aphotodiode or photomultiplier) mapped to the centre of the heat spot canbe used for detection of interactions, conformation etc. It isillustrated in the appended figures, particularly FIG. 27, how acapillary is used for thermo-optical characterization. A capillary maybe placed on a solid support/specimen holder/stage with good heatconducting properties. The solid support/specimen holder/stage may becooled or heated by a Peltier element. By using a Peltier element the“ambient temperature” of the solution can be adjusted. It isadvantageous to measure the protein conformation at differenttemperatures, to adjust thermophoresis of(bio)molecules/(nano)particles/(micro)beads to a value close to the signchange of thermophoresis (i.e. that a binding event changes thethermophoretic behaviour from accumulation at higher temperatures todepletion from higher temperatures). A cooling of the chamber allows toheat temperature sensitive molecules with a much higher laser power. Infurther embodiments valves are put at the end of the capillary toexclude any drift of the liquid inside the capillary. Often drift iscaused by evaporation at the end of a capillary.

However, it is also possible to provide the sample probe without achamber such as in form of a droplet, e.g. buffer droplet.

In some embodiments of the invention the fluorescence is detected withina range of from about 50 nm to 500 μm in direction of the laser beam.

In further embodiments the fluorescence is detected substantiallyperpendicular with respect to the laser light beam with a CCD camera.The second fluorescence detection is in some embodiments a spatialmeasurement of the fluorescence in dependence of the temperaturedistribution substantially perpendicular with respect to the laser lightbeam.

The appended figures show particular, non-limiting setups for devices inaccordance with the present invention in accordance with the presentinvention. These devices are particularly useful in measuringthermophoresis. Common to all of them is that fluorescence imaging andthe infrared laser focussing are performed through the same opticalunit, e.g. through the same objective. Particularly, an objective withvery low refraction of the IR light may be used. This is beneficialbecause a local spatial temperature profile on the micrometer scale isadvantageous and desired in context of the means and methods providedherein. High refraction of IR light by the objective may lead to a broadtemperature distribution with a high background temperature elevation.To solve this less advantageous effect, an objective with hightransmission in the IR region of the electromagnetic spectrum(preferably at 1200-1600 nm, i.e. corrected for IR radiation) may beused. An objective which comprises only a small number of lenses (i.e.objective with less correction for visible wavelengths) is herepreferred. As described herein, if a microfluidic chamber with a highaspect ratio (length/width) is used it may be advantageous to change theIR laser beam profile to an ellipsoidal form, to homogeneously heat thewhole cross section of the capillary. This allows, inter alia, highprecision measurements with a line camera, photodiode orphotomultiplier. A line camera provides merely line resolution along thecapillary and averages the spatial fluorescence of a whole cross section(width) of the capillary.

The photodiode or photomultiplier has no spatial resolution, but it ispositioned in a way to measure the fluorescence in the central heatedregion. Such a positioning is within the normal skills of the personskilled in the art. Both, line camera and photodiode combined with anellipsoidal illumination of the microfluidic chamber (i.e. capillary)are used in some embodiments of the invention for data acquisition.

Appended FIG. 23 illustrates a corresponding further embodiment, whereinthe simultaneous detection of two or more different fluorophores/markedparticles is documented. The different emission wavelength of the two ormore marked particles/molecules are splitted e.g. via a dichroic mirroror AOTF into two or more different directions. In this embodiment, onedetection channel can for example be used to measure the temperature viathe temperature dependent fluorescence of a dye, e.g. Cy5 at awavelength for example 680 nm+/−30 nm. In the other channel the meltingcurve of a marked particle/molecule can be recorded at a wavelength offor example 560 nm+/−30 nm. It also allows for the parallel detection ofparticles/molecules, e.g. different particles/molecules, with e.g.different luminescent or fluorescent markers.

An advantage of the present invention, is that, in contrast to the priorart, now particles, in particular and as exemplified, (bio)molecules or(nano- or micro-)particles or (micro)beads can bemeasured/determined/characterized by employing spatial temperaturedistributions with μm resolution.

Accordingly, with the means, methods and devices provided herein it is,inter alia, possible to measure, detect and/or verify biological,chemical or biophysical processes and/or to investigate, study and/orverify samples, like biological or pharmaceutical samples. Alsodiagnostic tests are feasible and are embodiments of this invention. Itis, inter alia, envisaged and feasible to measure the length of nucleicacid molecules (like DNA, RNA), to measure the melting features ofproteins or nucleic acid molecules, like, e.g. of double-stranded DNA ordouble-stranded RNA (dsDNA/dsRNA) or of hybrid nucleic acid molecules,like DNA/RNA hybrids, to measure and or analyze nucleic acid sequences,like the detection and/or measurement of Single Nucleotide Polymorphisms(SNPs) (see also appended figures, in particular FIG. 4) or to measurethe stability of nucleic acid molecules in correspondence and as afunction of their relative length; to measure and/or verify PCR endproducts, e.g. in general medical diagnostic, also in polar-bodydiagnostic, pre-implantation diagnostic, forensic analysis. Accordingly,it is evident for the skilled artisan that the means and methodsprovided in this invention are, particularly and non-limiting, useful inmeasurements and/or verifications wherein the length, size, affinitiesto other molecules/particles of a given particle/molecule is ofinterest. For example, the methods provided herein as well as thedevices are useful in the detection and measurement of length,temperature stability as well as melting points of nucleic acidmolecules and proteins. Therefore, it is within the scope of the presentinvention that, for example, (DNA-) primers and (DNA- or RNA-) probesare measured and or verified after or during their synthesis. Also themeasurement of nucleic acid molecules on templates, like DNA-chips isenvisaged. The term melting in context to this invention refers to thethermal denaturation of biomolecules, like nucleic acids (e.g. RNAs,DNAs) or proteins.

Also envisaged in context of the present invention is the measurement,detection and/or verification of mutations and genetic variations innucleic acid molecules, for example in form of single-strandconformational polymorphisms (SSCPs) or in form of restriction fragmentlength polymorphisms (RFLPs) and the like. The present invention alsoprovides for the possibility to analyze heteroduplexes. Heteroduplexesare generated by heat denaturation and reannealing of a mixture of e.g.wild type and mutant DNA molecules. In particular it is also possible tomeasure the effect of protein binding to a DNA molecule on the stabilityof the latter. Furthermore it is possible to measure the thermalstability of proteins and the effect of molecules (e.g. small molecules,drugs, drug candidates) on the thermal denaturation.

Also within the scope of the present invention is, e.g., the measurementof protein-protein interactions, like complex formations ofproteinaceous structures or of proteins or of fragments thereof. Suchmeasurements comprise, but are not limited to the measurement ofantibody-antigen binding reactions (also in form of single chainantibodies, antibody fragments, chromobodies and the like). Yet, theembodiments of the present invention are also related to the detectionand or measurement of dissociation events, like, e.g. the dissociationof protein complexes. Therefore, the invention is also useful in themeasurement, determination and/or verification of dissociation events,like in the measurement of the dissociation of proteinaceous complexes,e.g. antibody-antigen complexes and the like. The appended figures alsoshow that the means and methods provided herein are useful, for example,in the measurement of melting curves for nucleic acid molecules,proteins and corresponding analyses.

It is understood by the person skilled in the art that the term“modified microparticle/nanoparticle” as employed herein is not limitedto “microparticle/nanoparticle” per se but that the herein disclosedmeans, particles and materials may also be used and employed by usage ofother means, in particular, colloidal means, like foams, emulsions andsols.

As microparticles show a very strong thermophoresis, as illustrated inthe appended examples and figures, particularly in FIG. 33, they can beused as a carrier material for the detection and characterisation of forexample biomolecules, like proteins or nucleic acids. By usingmicroparticles, the thermophoresis signal of e.g. the biomolecules maybe enhanced. The attraction of a microparticle to an extreme value (e.g.the temperature maximum) of the spatial temperature distribution due tothermophoresis may be strong enough to trap the microparticles there (asillustrated in the appended examples and figures, particularly in FIG.34).

A microparticle is a particle with a characteristic length of less than1 mm and more than 100 nm without restriction of the material (e.g.coated or uncoated silica-/glass-/biodegradable particles,polystyrene-/coated-/flow cytometry-/PMMA-/melamine-/NIST particles,agarose particles, magnetic particles, coated or uncoated gold Particlesor silver Particles or other metals, transition metals, biologicalmaterials, semiconductors, organic and inorganic particles, fluorescentpolystyrene microspheres, non-fluorescent polystyrene microspheres,composite materials, liposomes, cells and the like).

A nanoparticle is a particle with a characteristic length of less than100 nm without restriction of the material (e.g. quantum dots,nanocrystals, nanowires, quantum wells).

Particles or beads according to this invention may be modified in such away that for example biomolecules, e.g. DNA, RNA or proteins, may beable to bind (in some embodiments specifically and/or covalently) to theparticles or beads. Therefore, within the scope of this invention is thethermo-optical analysis of characteristics of beads and/or particles andin particular of molecules attached to or linked to such beads orparticles. In particular, such molecules are biomolecules. Accordingly,the term “modified (micro)beads/(nano- or micro)particles”, inparticular, relates to beads or particles which comprise additionalmolecules to be analyzed or characterized (a non limiting example forthe case of nanoparticles is shown in the appended figures and examples,particularly in FIG. 34). Modified or non-modified microparticles/(nano-or micro)particles may be able to interact with otherparticles/molecules such as biomolecules (e.g. DNA, RNA or proteins) insolution. The skilled person understands that the thermophoreticproperties of the modified particles will change upon binding of thebiomolecules in solution to the biomolecules bound to the particle asmodification. Such an interaction may influence the force acting on the(modified) particle/molecule. By adjusting the IR-Laser irradiation, theresulting movement can be influenced in such a way that theparticle/bead is trapped. “Trapping” the particle/bead, in particularparticles/beads comprising biomolecules, means that the particle/beadstays within a certain position, only showing comparably lowfluctuations. These fluctuations are different from fluctuations basedon Brownian motions. When a biomolecule from solution binds to thebiomolecular-modified particle, the force acting on the particle/beadwill change due to a change of the thermophoretic properties, which mayresult in movement of the particle out of the certain position where theparticle/bead was trapped or/and in a change of the fluctuations of theparticle/bead. The method described here is termed “Thermooptical Trap”and is particularly useful in certain embodiments described herein. The“Thermooptical Trap” is also illustrated in the appended examples andfigures. Other synonyms for “Thermooptical Trap” are “Optothermal Trap”,“Thermophoretic Trap” as well as “Optothermal Tweezers” or“Thermophoretic Tweezers”.

The invention, accordingly, also relates to an optothermal trap. Theterms “thermo-optical”, “thermooptical”, “optothermal” and“opto-thermal” are used synonymously. This particular embodiment of theinvention illustrates that given targets, e.g. fluorescently labelledmodified beads/particles with a size of 100 nm up to several μm (e.g.polystyrene beads or silica beads) and lipid vesicles and cells areshowing a directed movement to the temperature maximum of a spatialtemperature distribution generated by applying IR-Laser radiation to anaqueous solution as illustrated in the appended figures, particularlyFIG. 39.

As shown in the appended example, the devices and the methods of thepresent invention may also be employed for thermophoretic trapping ofmolecules or particles, including lipid structures (like vesicles orliposomes) as well as cellular components of even cells. It is alsoenvisaged that the devices and, methods of the present invention is usedfor thermophoretic trapping of cells or cellular components, like cellnuclei, chromosomes, mitochondria, chloroplasts and the like. Thethermophoretic trapping as shown herein is particular useful forstudying interactions of e.g. proteins (e.g. with other proteins, forexample antibody-antigen interactions and the like), for studyingtransport events across membranes (e.g. vesicles or liposomes), fordetermination of activity of membrane proteins comprised in biologicalmembranes/vesicles/liposomes, like ion pumps, membrane transporters andthe like. Also, the mere presence of molecules, particles, liposomes,vesicles, beads, cells or cellular components in said solution can bedetected and/or analyzed by use of the herein disclosed thermophoretictrapping devices and methods. Thermophoretically trapped molecules,particles, vesicles, beads, cells or cellular components and the likecan be transported and moved within the analyzing solution (see alsoappended figures). It is envisaged that thermophoretically trappedmolecules, particles, liposomes, vesicles, beads, cells or cellularcomponents be exposed to different buffer solutions for certainapplications, i.e. buffer around the trapped molecules, particles,vesicles, beads, cells or cellular components etc. may be exchanged andcorresponding measurements can be carried out. Further embodiments incontext of the thermophoretic trapping in accordance with this inventionare also provided in the appended examples. It is evident for the personskilled in the art that the concepts of thermophoresis as disclosedherein can also be employed for example in sorting of vesicles, cellularcomponents (like e.g. mitochondria, chloroplasts, nuclei, chromosomes)or even whole cells. Accordingly, the present invention also providesfor a method to thermo-optically trap molecules, particles, vesicles,beads, liposomes, cells or cellular components etc., said methodcomprising the steps of providing a sample probe with (preferablymarked) molecules, particles, vesicles, beads, liposomes, cells orcellular components; irradiating a laser light beam into the solution toobtain a spatial temperature distribution in the irradiated laser lightbeam; optionally detecting the (preferably marked) molecules, particles,vesicles, beads, cells or cellular components; and trapping the(preferably marked) molecules, particles, vesicles, liposomes, beads,cells or cellular components in accordance to the thermophoreticmobility of said molecules, particles, vesicles, beads, cells orcellular components. For example, the (preferably marked) molecules,particles, vesicles, beads, cells or cellular components can be trappedin center of the laser-generated heat spot (in particular whenthermophoretic mobility of the trapped molecules, particles, vesicles,liposomes, beads, cells or cellular components etc is negative).However, the (preferably marked) molecules, particles, vesicles, beads,cells or cellular components can also be trapped in a (global or local)temperature minimum, in particular, when the thermophoretic mobility ofthe trapped molecules, particles, vesicles, beads, cells or cellularcomponents etc is positive. The person skilled in the art is aware thatthe term “thermophoretic mobility”, DT refers to a coefficient whichrelates the velocity (v) of a given molecule/particle/bead etc to thetemperature gradient (∇T), according to v=−DT∇T.

The embodiments provided herein above in context of the method formeasurement of thermo-optical characteristics of particles/moleculesetc. in a solution apply to the method of thermo-optically trappingmolecules, particles, vesicles, beads, liposomes, cells or cellularcomponents, etc mutatis mutandis. Also devices for the thermo-opticallytrapping are provided herein and are also illustrated in the appendedfigures, for example the device as shown in appended FIG. 19 or 24. Acorresponding device comprises, accordingly, an IR laser for irradiatinga laser beam into the solution containing the (preferably marked)molecules, particles, vesicles, beads, liposomes, cells or cellularcomponents to be trapped, to obtain a spatial temperature distributionin said solution around the irradiated laser light beam. Said device forthermo-optically trapping (preferably marked) molecules, particles,vesicles, beads, liposomes, cells, cellular components, etc, maytherefore comprise: (a) a receiving means for receiving (optionallymarked) molecules, particles, vesicles, beads, cells, cellularcomponents, etc within a solution; (b) (optionally) means forfluorescently exciting the marked particles; (c) (optionally) means fordetecting the excited fluorescence in said solution; and (d) an IR laserfor irradiating a laser light beam into the solution to obtain a spatialtemperature distribution in the solution around the irradiated laserlight beam.

The movement of molecules, particles, vesicles, liposomes, beads, cells,cellular components, etc. can be described by a thermophoretic forceacting on the particle. Presuming thermodynamic equilibrium, this forcemay be derived from the Gibbs free enthalpy at constant pressure:F=−½*S _(T) *k _(B)*grad(T ²)

Where S_(T) is the Soret coefficient and k_(B) the Boltzmann constant.

The temperature T is a function of x and y: T=T(x,y). For example, in aradial symmetric geometry like the spatial temperature distributiongenerated by a focused IR-laser (as illustrated in the appended figures,particularly in FIG. 3 a) it can easily be seen that the force canattract a particle to the maximum of the spatial temperaturedistribution. If S_(T)<0 this results in a force trapping the giventarget particle/bead, e.g. a silica microparticle, preferably a coatedsilica microparticle, at the maximum (local or global) of thetemperature distribution.

In contrast to the optical tweezers/optical trapping known from the art,the thermo-optical trap according to the present invention is based ondifferent principles. Instead of using an electromagnetic field gradientas used for optical tweezing, a temperature gradient is used to trap,move and control a particle according to the present invention.Therefore, no sophisticated confocal optic is necessary in someembodiments of the thermo-optical trap. Using temperature gradients alsoallows for attraction of molecules from distances of 1 μm to severalhundred micrometer distance to the laser focus, depending on the widthof the temperature gradient (e.g. the width of the IR laser focus).Compared to the thermo-optical trap, the catchment region for opticaltweezers is very narrow on the order of a few micrometer.

As shown in the appended Example 1, the Soret coefficient S_(T) is afunction of the surface area A of the target particle, e.g. the bead,the quadratic effective charge σ_(eff) and particle-area-specifichydration entropy s_(hyd). So the thermophoretic force will also beproportional to this particle properties.

If one of this properties changes (preferably the effective charge orthe hydration entropy), the trapping force changes also. If the trappingforce/the trapping potential changes, the fluctuations (as alsoillustrated in the appended FIG. 32) also change By recording thefluctuations of the particle, a change in the thermophoretic propertiesof the particle can be detected and so the binding of for examplebiomolecules to this particle/bead can be detected.

In case of a particle/bead, e.g. a silica microparticle, preferably acoated silica microparticle, more preferably a silica microparticlecoated with special groups on its surface (e.g. proteins). These groupsmay be able to specifically bind to proteins, antibodies, smallmolecules, DNA, RNA etc. If there is a binding of one of this species tothe specific group on the bead/particle, the properties (for example thesurface A) of the particle, e.g. bead changes, which may result in adifferent S_(T) and thus to a different thermophoretic force F (e.g. thesign/direction of the force may change).

A particular embodiment of the invention relates to the measurement ofthe fluctuations of such a given target particle/molecule in the maximumof the temperature distribution by detecting its position viameasurement of fluorescence or the like. When the particle/molecule istrapped in the temperature distribution, the solution around it can beeasily exchanged with a solution containing non-labelled or labelledmolecules which are envisaged to bind to the “trapped” particle, e.g.when modified in a way that allows the specific binding (e.g. byantibodies). A binding event may be detected by a change in theamplitude of fluctuations (i.e. the potential in which the particle istrapped changes due to the binding of molecules to the particle'ssurface). By detecting the time dependent characteristics of thevariation in amplitude, a binding kinetic can be measured andestablished.

In a further particular embodiment of the invention, the change in signof S_(T) of the target particle may be exploited. If S_(T)<0 a particle,e.g. a bead or a fluorescent silica microparticle, with specific bindinggroups/sites is trapped at the maximum temperature in the spatialtemperature distribution. If S_(T) of the target particle changes itssign upon e.g. crowding or linking of the binding groups with moleculese.g. small molecules, the target particle is forced away from themaximum of the spatial temperature distribution and the particle, e.g.bead, experiences repulsion instead of attraction, resulting in adetectable qualitative change of the behaviour of the target particle.For example if the force changes from attraction to repulsion, theparticle may move away from the maximum of the spatial temperaturedistribution. Thereby, molecules, e.g. small molecules, binding to saidbinding groups at the surface of the particle/bead, may be detected. Thebinding of molecules, e.g. small molecules, can easily be measured justby simple particle tracking methods. The particle can be brought toconditions close to a sign change by changing the buffer conditions(e.g. salt concentration) the temperature of the solution or by specificmodifications with e.g. hydrophobic or charged molecules. It is evidentthat the term “target particle” or “target bead” also relates in thisembodiment to correspondingly modified particles and beads, likeparticles/beads comprising or linked to biomolecules or particles/beadscoated with such biomolecules. Therefore, the above recited embodimentsfor “particles/beads” and in particular modified particles/beads applyhere mutatis mutandis.

The force acting on the target particle, e.g. a silica microparticlecoated with special groups, can be measured by tracking its position byan appropriate method like fluorescence (if the particle isfluorescently excitable), phase contrast, interference, farfield imagingetc.

It is also possible to apply a second force onto the target particle,e.g. a specifically coated magnetic microparticle, in a way that theresulting force is a superposition of the thermophoretic force and thesecond force (e.g. a magnetic force). The second force can be forexample a magnetic force (for magnetic particles) or an electric force(for charged particles) or a hydrodynamic force or another optical forcelike it is generated by an optical tweezers.

Then the resulting superposition of forces may then be measured, forexample by measuring the fluctuations of the particle (as alsoillustrated in the appended FIG. 32). This superposition may be used toincrease the sensitivity of the method for example by usingcounteracting forces in such a way that a small change in one of theproperties of the target particle/bead, will result in a movement of thetarget particle/bead, whereas without a change in the properties of theparticle/bead, the particle/bead stays at the same place. The increaseof sensitivity may be due to the possible minute adjustability of thesecond force (e.g. a magnetic force).

The “Thermooptical Trap” can also be used to move a targetparticle/molecule, e.g. a bead/particle, in two dimension perpendicularto the axis of the incident IR-Laser radiation. If the focus of theIR-Laser is moved for example by use of galvanic mirrors or an acousticoptic deflector (AOD) the resulting maximum of the spatial temperaturedistribution is also moved and so is the target particle/molecule/bead.Or vice versa, the chamber may be moved and the IR-Laser focus is keptfixed (as also illustrated in the appended figures, particularly FIG.34).

Using a variety of IR-Laser focal spots lots ofparticles/beads/molecules can be moved simultaneously, giving theopportunity of multiplexing and also of combining different targetparticles, e.g. specifically coated microparticles, with each other. Soif there is one target particle with an antibody and another targetparticle with the corresponding antigen, the target particles can bemoved by two IR-Laser foci until they are in contact and the antibodybinds to the antigen. In this way the target particles/molecules bind toeach other and a composition of particles may be created.

In accordance with this invention, it is also possible to generate aninterference pattern of the IR-Laser radiation, resulting in a spatialgrating of temperature maxima. With this spatial grating of spatialtemperature distribution target particles/molecules can be trapped andthey can also be moved by moving the interference pattern.

As also demonstrated in the appended figures, the present invention isparticularly useful in the determination of single or double strandednucleic acid molecules (see, e.g. appended FIG. 5). This allows, interalia, to determine in a given probe/sample whether it comprises singleand/or double stranded nucleic acid molecules. This is in particularrelevant in cases when it has to be determined whether a givenbiological sample comprises, e.g. viral nucleic acids, like singlestranded DNA or single stranded RNA.

As documented herein, one embodiment of the present invention is basedon the fact that with the means and methods of this invention it ispossible to measure, in very short time intervals, inter- as well asintra-molecular interactions. In the first illustrative embodiment ofthe invention as described herein, a thermo-optical method is disclosedthat allows for the detection of a broad temperature range (in a givenprobe/sample) at the same time, whereby said “same time” is a time rangeof about 1 ms to 250 ms, in particular 80 ms to 180 ms and, asexemplified at 150 ms, yet, at the most at 250 ms. This firstillustrative embodiment of the invention is not based on or related tothermophoresis. In contrast, thermophoresis is, to a large extendexcluded. The first embodiment is, e.g. related to the determination ofmelting curves, e.g. the determination of DNA and protein melting(point) curve(s). A non-limiting example of this first embodiment is thedetermination/measurement of single nucleotide polymorphism, based asprovided in appended FIG. 4. The “melting point” is defined by 50%dissociated molecules. It is evident herein that the disclosed method asprovided in the first embodiment is not limited to the determination ofmelting points of DNA molecules.

In the second illustrative embodiment of the invention, thermophoresisor thermophoretic effects play a role, inter alia, in a pre-determinedtime of about 0.5 second to about 250 seconds, preferably about 1 secondto about 150 seconds, more preferably about 5 seconds to about 100seconds, more preferably about 5 seconds to about 80 seconds morepreferably about 5 seconds to about 50 seconds and even more preferablyin about 5 seconds to about 40 seconds, concentration changes within aspatial temperature distribution are measured and/or detected. Here,concentration changes and not structural changes of theparticles/molecules to be characterized in accordance with the inventivemethods are measured/detected. Structural changes in this context arerelated to thermal denaturation mentioned in the first embodiment. Thesecond illustrative embodiment illustrates that conformational changesand changes in surface (like size and chemistry) and interactions may bemeasured by thermo-optical characterization because the thermophoreticproperties are altered. Also, thermo-optical “trapping” devices areillustrative for this embodiment. Corresponding illustrations of theusefulness of this embodiment of the invention are also illustrated inthe appended examples, e.g. the determination of hydrodynamic radius andinteraction between proteins, the detection of interactions betweenbiomolecules and discrimination of nucleic acids by size, the detectionof binding of molecules to particles, the investigation of conformation,structure and surface of (bio)molecules, the detection of conformationalchanges, like folding/unfolding of biomolecules, the trapping ofparticles (e.g. the trapping of vesicular structures or lipids) or(bio)molecules, and the detection of covalent and non-covalentmodifications of particles.

It is documented and exemplified herein below that, e.g. thethermophoresis of nucleic acids (in particular of DNA) is length/sizedependent and the means and methods provided herein allow for thedetermination and elucidation of single versus double stranded DNA aswell as the determination also of small nucleic acids up to e.g. 100,300, 1000, or 5000 nucleotides or base pairs. A non-limiting example isillustrated in appended FIG. 5, wherein mobility in a temperaturegradient is measured by the means and methods provided herein. Here itis shown that in particular the second embodiment of this inventionallows for the distinguishing verification between length/size (in theparticular example 20 mer versus 50 mer) and/or “strandness” of nucleicacid molecules (in the particular example single stranded DNA versusdouble-stranded DNA). Again, also this second illustrative embodiment ofthe present invention is not-limited to the detection of short DNA orthe determination of double- or single-stranded nucleic acid molecules.Also interactions between particles/molecules, conformations,hydrodynamic radii, binding kinetics and stabilities ofparticles/molecules, e.g. proteins, nucleic acids (e.g. DNA, RNA, PNA,LNA), nanoparticles, beads, particularly microbeads, lipids, liposomes,vesicles, cells, biopolymers (hyaluronic acid, alginate and alike),two-dimensional lipid sheets, inorganic substances (e.g.carbon-nanotubes, buckyballs, etc), Poly-ethylenglycol (PEG) may bemeasured. The molecules mentioned above show e.g. differences intemperature stability. Illustrative molecule specific temperature rangesfor the measurement of respective thermo-optical properties are given intable 1.

The examples for uses of the means, methods and devices disclosed hereinare not to be considered to be limiting and illustrate the invention. Inparticular, the present invention and its corresponding means andmethods are not limited to the use of the detection, measurement and/orverification of biomolecules, like nucleic acids orproteins/proteinaceous structures. As is evident from the invention asdisclosed herein, also any temperature-sensitive system can be adoptedto the methods and devices disclosed herein.

It is, e.g. feasible to measure also chemical reactions, like inorganicor organic reactions.

The person skilled in the art is aware that the invention as disclosedherein is only restricted by the fact that the reaction to be measured,detected, verified and or assessed has to take place in an solution thatcan be heated, in particular heated optically.

In some embodiments the device according to the present invention isbased on a fluorescence microscopy setup with an excitation means, e.g.a Light Emitting Diode (LED) for excitation, an excitation/emissionfilterset, a specimen holder for a microfluidic chamber and a fast CCDCamera for spatial resolved recording of the fluorescence intensity.Such a fluorescence microscopy setup is well established in life scienceand other areas. According to the present invention, such a common setupis extended by an infrared (IR) laser whose radiation is focused. Thelaser may be arranged below the specimen holder such that the radiationis focused from below the specimen holder into the microfluidic chamberby an IR corrected lens (as illustrated in the appended figures,particularly FIG. 1). However, the laser, the detection means and theexcitation means may be arranged on one common side of the specimenholder, e.g. below the specimen holder as depicted for example in FIG.2. In one embodiment, the specimen holder is attached to the objective.Such a setup avoids relative movements of the specimen holder withrespect to the objective. According to a further embodiment, it ispossible to move the laser freely in the object plane by using twovoltage driven infrared mirrors. It is further advantageous to use thinliquid films (approx. 1 μm to 500 μm, preferably 1 μm to 50 μm, morepreferably 1 μm to 20 μm, even more preferably 1 μm to 10 μm), e.g. inthin liquid chambers, of biomolecule solutions with local coherent IRradiation. However, the method of the present invention is not limitedto thin liquid chambers. An extension to μl-drops or nl-drops of aqueoussolutions, capillaries and micro-well plates is possible as illustratedin the appended figures, e.g. FIGS. 2 and 16 to 24. According to afurther embodiment, the infrared heating and fluorescence detection arerealised through the same objective, which makes the setup much moreflexible and compact (see e.g. FIGS. 2 and 16 to 24). The use of oneobjective to focus both electromagnetic radiation from the infrared andvisible part of the spectrum comprises the necessity that the objectivedoes both with high optical quality. In particular the infraredradiation must not exhibit a strong dispersion by the objective. Strongdispersion would lead to a high temperature offset and a comparablystrong temperature gradient at distances from the heated centre. Thissituation is in some embodiments described here avoided. Strongdispersion increases the time of measurement and decreases theprecision. Without being limited by theory, this may be due to anincrease of the length scale where thermophoresis is strong whereby thesystem needs more time to reach steady state. The second effect may bedue to the fact that the nonlinear bleaching correction is only preciseif thermophoresis is negligible at greater distance to the heat spot.This is achieved when diffraction of the infrared radiation is low.Accordingly and in accordance with this invention, only one direction inspace may be used for detection and manipulation. The described methodand the herein disclosed devices may be integrated into establishedinstruments and high throughput systems.

Water shows a strong absorption of radiation in the infrared regimelarger than 1200 nm. The absorbed energy is converted into heat.Coherent IR LASER and IR optics allow the creation of a very high powerdensity of infrared radiation in solution. By controlling the LASERoptics the LASER focus can be moved and changed. This includes opticswhich change the aspect ratio of the radial symmetric laser beam toproduce a line shaped focus. This is in particular useful ifmeasurements are conducted in a capillary. Since the whole cross sectionis heated homogeneously, a spatial temperature profile exist only alongthe length of the capillary. A temperature gradient in only onedirection of space increases the precision of the measurement since theall pixels with same distance from the heated centre can be averaged. Inparticular this allows the use of a CCD camera with a single line ofpixels. In this case the integration of fluorescence is obtained byhardware. By using a photodiode or photomultiplier the fluorescence froma finite volume element (i.e. from the centre of the heat spot/line) ismeasured, without any spatial resolution. A spatial resolution offluorescence detection is only necessary in cases were the hydrodynamicradius is the thermo-optic property of interest. The optical heatingtechnique allows the creation of broad temperature distributions andstrong temperature gradients on the micron scale. At the position of theLASER focus there is the highest temperature. This upper temperaturelimit may be adjusted by controlling the power of the LASER and theshape of the laser focus. With increasing distance to the laser focusthe temperature of an aqueous solution is decreasing due to thermalconductivity. The lower limit of the temperature may be set by thetemperature of the surrounding chamber material. This material can becooled down, e.g. up to 0° C. In this way it is possible to generate atemperature distribution containing all temperatures between 100° C.(with high laser power) at the laser focus and 0° C. at greaterdistances to the heat spot.

With the method of the present invention it is possible to heat andanalyze solutions, particularly aqueous solutions in a thermo-opticalway. There is no need for heat conducting materials like heattransducers from a heating element (copper wire, Peltiers etc.). Thesolution itself is directly heated by the LASER light. Because the laserfocussing is only diffraction limited, temperature distributionsspanning all temperatures between 0° C. and 100° C. (the complete liquidphase of water) can be observed simultaneously on a length scale of afew hundred micrometers.

With the method of the present invention, measurements are 3000-10000times faster than the fastest available measurement systems known in theart. The method of the present invention allows to obtain alltemperatures between 0° C. and 100° C. at the same time because aspatial temperature distribution is used. The temperature is not createdby contact with a heating element, but within the sample itself. Byusing the infrared scanning optics, arbitrary two dimensionaltemperature patterns can be created in solution. This way, anystructuring of the surface is obsolete. In addition, all materialstransparent for radiation in the infrared can be used to build amicrofluidic measurement chamber (glass, sapphire, plastic, silicon,crystals). In addition, the method of the present invention can also beused to create temperature distributions in the aqueous solution near asurface. Because of the continuity of temperature the surface alsoadopts a temperature distribution. Therefore it is possible to heat thesurface as well as the solution. A possible application is the analysisof DNA-microarrays. Temperature gradients close to a surface may be usedto move molecules toward a surface or away from a surface. These localconcentration changes can be measured precisely by total internalreflection fluorescence (TIRF) system shown in the appended figures,particularly FIGS. 24 and 36, or any optical system (1) capable of TIRF.This thermophoretic motion in the direction of the incident laser lightcan be used to direct molecules into a microfluic structure in order tocapture and/or concentrate them. Therefore, temperature gradientsgenerated according to the present invention can also be used to captureand/or concentrate molecules/particles. The capture and concentration ofmolecules/particles is dependent on their thermo-optical properties(e.g. the sign of the thermophoretic effect).

One way to suppress secondary effects related to inhomogeneoustemperatures in solution can be suppressed by choosing the rightmicrofluidic chamber geometry. For example convection is dealt with byusing only a thin sheet of liquid. This also means that it isadvantageous for reproducible and precise measurements that the heightof the thin sheet of liquid does not vary from measurement tomeasurement. The speed of the convective flow, which is accompanied bythe spatial temperature distribution depends quadratically on the heightof the liquid sheet. This nonlinearity means that slight changes inchamber height lead to comparatively strong changes in the speed of theconvective flow which in turn effects the concentration and temperatureprofile in a very complicated way. Therefore experiments are preferablyperformed in microfluidic measurement chambers of defined height (e.g.capillaries). Since in accordance with the means and methods of thisinvention temperature is generated herein by the generation of heat dueto the absorption process, the constant height is also advantageous toobtain reproducible temperature distributions. Differences in heightwould lead to deviations due to differences in the amount of energyabsorbed and because of differences in the volume/surface ratio. Thisratio determines the rate of heat transfer to the surrounding andtherefore also the temperature distribution in solution. Thereproducibility of the temperature profile determines the maximalpossible measurement precision.

Another way is to measure even faster to avoid disturbances byconvection opening the possibility to measure in single droplets ormicro-well plates (thicker sheet of liquid). Since the IR laser isabsorbed on a length scale of 300 μm (1/e) thin samples, e.g., thinchambers are heated homogeneously in the z-direction (height).

Accordingly, the present invention provides an improved method tomeasure thermo-optically characteristics of particles/molecules in asolution with the steps of (a) providing a sample probe with markedparticles/molecules in a solution; (b) exciting fluorescently saidmarked particles and firstly detecting fluorescence of said excitedparticles/molecules; (c) irradiating a laser light beam into thesolution to obtain a spatial temperature distribution in the solutionaround the irradiated laser light beam; (d) detecting secondly afluorescence of the particles/molecules in the solution at apredetermined time after irradiation of the laser into the solution hasbeen started, and characterizing the particles/molecules based on saidtwo detections.

In one embodiment the predetermined time is within the range of from 1ms to 250 ms. Preferably the detection time is in the range of from 1 msto 50 ms. In a particular embodiment, the laser beam is defocused suchthat a temperature gradient within the temperature distribution is inthe range of from 0.0 to 2 K/μm, preferably from 0.0 to 5 K/μm.Preferably the laser beam is irradiated through an optical element intothe solution. In a particularly embodiment the optical element is asingle lens. In a particular embodiment of the invention, the method isfurther comprising the step of measuring the temperature distribution inthe solution around the irradiated beam with a temperature sensitivedye. The temperature distribution may be determined based on detectedfluorescence of the temperature sensitive dye, wherein the solutioncomprising said temperature sensitive dye is heated by the irradiatedlaser beam and the fluorescence spatial fluorescence intensity ismeasured substantially perpendicular around the laser beam. In a furtherembodiment the predetermined time is within the range of from 0.5 s to250 s. Preferably in said predetermined time concentration change(s)within the spatial temperature distribution in the solution due tothermophoretic effects and such (an) concentration change(s) is(are)detected by a change of the distribution of fluorescence. In someembodiments the laser beam is focused such that a temperature gradientwithin the temperature distribution is achieved in the range of from0.001 to 10 K/μm. In a further embodiment of the invention fluorescenceis detected with a CCD camera. In some embodiments, the brightness ofsaid fluorescence is detected with a photodiode or a single pixel withthe CCD in the centre of the laser beam. In further embodiments theparticles are biomolecules and/or nanoparticles and/or microbeads and/orcombinations thereof. In particular embodiments, the laser light iswithin the range of from 1200 nm to 2000 nm. Preferably the laser is ahigh power laser within the range of from 0.1 W to 10 W, more preferablyof from 0.1 W to 10 W, even more preferably from 4 W to 6 W. In someembodiments, the solution is an aqueous solution with an particleconcentration within the range of from 1 atto Molar (single ParticleMicrobeads) to 1 M, preferably from 1 atto Molar to 100 μMolar.Particularly, the solution is a saline solution with concentrations inthe range of from 0 to 1 M. Preferably, the spatial temperaturedistribution is between 0.1° C. and 100° C. In preferred embodiments,the temperature gradient is created within 0.1 μm to 500 μm in diameteraround the laser beam. The irradiation of the laser and the detection ofthe fluorescence is in a preferred embodiment of the invention conductedfrom the same side with respect to the sample probe. Preferably, thesolution is provided with a thickness in direction of the laser lightbeam from 1 μm to 500 μm. In particular embodiments, the fluorescence isdetected within a range of from 1 nm to 500 μm, particularly from 50 nmto 500 μm in direction of the laser beam. Preferably, the fluorescenceis detected substantially perpendicular with respect to the laser lightbeam with a CCD camera. More preferably the second fluorescencedetection is spatial measurement of the fluorescence in dependence ofthe temperature distribution substantially perpendicular with respect tothe laser light beam. In preferred embodiments, the sample solution isin a capillary.

The present invention also provides a device for measuringthermo-optically characteristics of particles in a solution as describedin any of the above embodiments, wherein the device comprises: areceiving means for receiving marked particles within a solution; meansfor fluorescently exciting the marked particles; means for detecting theexcited fluorescence in said solution; a laser for irradiating a laserlight beam into the solution to obtain a spatial temperaturedistribution in the solution around the irradiated laser light beam. Insome embodiments, the means for fluorescently exciting the markedparticles is a LED. Preferably, the laser is a high power laser withinthe range of from 0.1 W to 10 W, preferably 1 W to 10 W, more preferablyfrom 4 W to 6 W. In a particular embodiment, the laser and the means fordetecting the excited fluorescence are arranged on the same side withrespect to the receiving means. In a more preferred embodiment, thedevice further comprises an optic for magnifying the detected region. Inparticular embodiments; the device further comprises an optic forfocusing or defocusing the laser beam. Preferably the optic is a singlelens. In preferred embodiments the detecting means is a CCD camera. Insome preferred embodiment, the CCD camera is a line CCD camera. Inparticular embodiments, the detection is one-dimensional along thelength of a capillary. In another particular embodiment, the detectingmeans is a photo diode.

The present invention also relates to the use of the methods and thedevices described in any of the above embodiments for detecting and/ormeasuring the characteristics of particles and/or molecules in solution.The molecules to be detected, measured or characterized in accordancewith this invention may also be drug candidates.

In particular embodiments of the invention, the characteristics to bedetected or measured in accordance with this invention are selected fromthe group of stability, length, size, conformation, charge, interaction,complex formation and chemical modification of particles. In preferredembodiments, the particles to be measured are selected from the groupconsisting of (a) molecule, biomolecule(s), nanoparticles, beads,microbeads, (an) organic substance(s), (an) inorganic substance(s)and/or combinations of these. Preferably said particle is selected fromthe group consisting of (a) (bio)molecule(s), nanoparticles,microparticles, microbeads, (an) organic substance(s), (an) inorganicsubstance(s) and/or combinations of these. More preferably, the(bio)molecule is selected from the group consisting of (a) protein(s),(a) peptide(s), (a) nucleic acid(s) (e.g. RNA (e.g. mRNA, tRNA, rRNA,snRNA, siRNA, miRNA), DNA), (an) RNA aptamer(s), (an)antibody/antibodies (or fragments or derivatives thereof), (a)protein-nucleic acid fusion molecule(s), (a) PNA(s), (a) locked DNA(s)(LNAs) and (a) biopolymer(s) (sugar polymer, hyaluronic acids, alginate,etc.). Also intra- or inter-molecular interactions, e.g. proteinfolding/unfolding, are within the scope of this embodiment.

A particularly preferred embodiment of the present invention relates toa method to measure thermo-optically the physical, chemical orbiological characteristics of particles/molecules in a solution with thesteps of (a) providing a sample probe with marked particles/molecules ina solution in a capillary; (b) exciting fluorescently said markedparticles/molecules and firstly detecting fluorescence of said excitedparticles/molecules one-dimensionally along the length of the capillary;(c) irradiating a laser light beam into the solution to obtain a lineartemperature distribution in the solution around the irradiated laserlight beam along the length of the capillary; and (d) detecting secondlya fluorescence of the particles/molecules in the solution at apredetermined time after irradiation of the laser into the solution hasbeen started, and characterizing the particles based on said twodetections.

The present invention also provides a particular device for measuringthermo-optically the physical, chemical or biological characteristics ofparticles/molecules in a solution as described in any of the aboveembodiments, wherein the device comprises: a capillary for receivingmarked particles/molecules within a solution; means for fluorescentlyexciting the marked particles/molecules; means for detecting the excitedfluorescence in said solution one-dimensionally along the length of saidcapillary; a laser for irradiating a laser light beam into the solutionto obtain a linear temperature distribution in the solution around theirradiated laser light beam.

The device according to the present invention with a fluorescencemicroscopy and local infrared laser heating may also be used to measurethe effect of temperature gradients (i.e. temperature inhomogeneities)on dissolved molecules (see second embodiment of the present invention).Almost all dissolved molecules start to move in a temperature gradient,either to hot or cold regions. This effect is called thermophoresis orSoret effect and is known for 150 years. But the mechanism of moleculemovement in liquids stayed unclear. A mayor step towards a theoreticalunderstanding of thermophoresis in liquids has been done recently.

With the method and the device according to the invention, thestability, conformation, size and/or length of molecules, in particularbiomolecules may be characterized and/or determined. The interaction of(bio)molecules with other molecules or particles, e.g. further(bio)molecules, nanoparticles or beads, e.g. microbeads is characterizedin particular embodiments of the invention. The molecules to be analyzedmay also be linked (e.g. covalently or non-covalently) to beads orparticles, e.g. the beads or particles may be coated with molecules(e.g. biomolecules) to be analyzed, characterized in accordance withthis invention.

In the following two illustrative methods according to the presentinvention which rely on a very similar measurement protocol but analyzevery different molecule parameters will be discussed. Only in the methodof the second illustrative embodiment of the present invention thethermophoretic motion of particles is used. In the method of the firstillustrative embodiment this effect has to be excluded. Furthermore,particular embodiments of the present invention will be explained indetail with reference to the figures and with reference to the appendeddetailed examples. Said references to the figures and examples are notconsidered to be limiting.

First Illustrative Embodiment of the Invention

The method according to a first illustrative embodiment is in particularuseful in a temperature stability measurement of molecules, inparticular biomolecules. However, it is again of note that the means andmethods provided herein are not limited to the detection, verificationand/or measurement of biomolecules. The method described and illustratedas a non-limiting example, in the following allows, e.g., themeasurement of melting temperatures (FIG. 3) (stability, thermodynamicparameters like dS (change in entropy), dH (change in enthalpy) and dG(change in Gibbs free energy)) of biomolecules (proteins, doublestranded (ds)RNA, dsDNA were one nucleic acid strand could also be boundto a (nano)particle, microbead, surface etc). With said methodmeasurements of melting curves of dsDNA and DNA hairpins have beenconducted. The results are very well comparable to respective literaturevalues. As mentioned herein above, the present invention is inparticular useful in the measurement of biomolecules in general.Illustratively, it is shown hereon that e.g. SNPs (single nucleotidepolymorphisms) in (short) DNA strands can easily be detected (see alsoFIG. 4).

A particular embodiment of the first illustrative method will beexplained in the following. Nucleic acids with a fluorescence tag aregiven into a thin microfluidic chamber (i.e. e.g. 40 μm, 20 μm, 10 μm,or 5 μm, preferably 20 μm). The modification of nucleic acids with tags,like fluorescent tags is a well established technique which is broadlyused. Before heating starts, the fluorescence is observed to determinethe fluorescence level of 100% not melted molecules. It is importantthat the used fluorescence tag reacts on the melting of the two DNAstrands (or RNA strands or protein structure, or the fluorescence of ananoparticle reacts on whether ssDNA/RNA or dsDNA/RNA are bound to it).This can be realized by using e.g. fluorophore/quencher pair(Donor/Quencher pair particularly Donor/Acceptor pair: Energy Transfer(ET), e.g. Resonance Energy Transfer (RET), particularly FluorescenceResonance Energy Transfer (FRET)) or by the dissociation of anintercalating nucleic acid stain (e.g. SYBR Green/POPO/YOYO) or aprotein stain (e.g. SYPRO Orange (Invitrogen)). In case of e.g.gold-nanoparticles, fluorescence changes by changing of index ofdiffraction by DNA binding. The laser coupled to the microscope isdefocused and adjusted in a way that the temperature gradients are lowenough to decrease the thermophoretic particle drift to negligiblevalues. At the same time focusing must be tight enough to reachtemperature high enough for melting of the molecules. The measurementsis in some particular embodiments performed with high temporalresolution in the microsecond range, since the measurement has to beperformed in a time span were thermophoretic motion is still negligiblebut the heating process of the microfluidic chamber is completed. Themeasurement is strongly dependent on the experimental conditions. Thedata necessary for determining the melting temperature are typicallyobtained within 200 ms, within 150 ms, within 100 ms or within 50 ms,preferably within 150 ms. This is very surprising and supports the gistof this invention. Even shorter time spans are possible. In context ofthis invention, e.g. for a qualitative discrimination of differentspecies or nucleic acids. A first image is taken before the IR laser isturned on to obtain the fluorescence level of 100% not melted molecules.A second image is taken 100 ms, preferably 50 ms, more preferably 40 msafter the laser is turned on (were the chamber has reached its steadystate temperature). These two images contain all necessary information.The first image contains the fluorescence of the not melted molecule.The image taken while the laser is turned on allows the observance ofthe percentage of melted molecules at all different temperaturessimultaneously, ranging from 0% far away from the heat spot (cold) to100% in the centre of the heat spot (hot). From an independentmeasurement the temperatures of all pixels in the melting experiment areknown. Plotting the percentage of i.e. melted DNA strands vs.temperature allows to determine the stability of the molecule (i.e. themelting temperature) and to derive the thermodynamic parameters.

In summary, the first illustrative embodiment of the present inventionconnects the measurement of a spatial temperature distribution on themicrometer scale with the measurement of temperature dependentchemical/biochemical reactions and temperature dependent interactionsbetween molecules/biomolecules/nanocrystals/microbeads. For themeasurements the absolute temperatures not the temperature gradients areimportant. One can consider each detection volume (mapped onto aCCD-camera-pixel) as an microreactor. For the measurement it is veryimportant that every molecule stays in this microreactor during the timeof measurement. Therefore the measurement has to be as fast as toprevent thermophoresis and convection to move the particle out of thearea addressed as microreactor. This requirement is satisfied withmeasurement time of 150 ms.

With the method and the device according to the first illustrativeembodiment one is able to discriminate between dsDNA molecules with asingle nucleotide mismatch (SNP, single nucleotide polymorphism), aswell as salt dependent and length dependent differences in stability.The differences in stabilities can also be measured for dsRNA, DNA andRNA hairpins (ssDNA/RNA base pairing with themselves) and proteins. Alsothe influence of different aqueous buffers systems to the stability canbe measured (pH, salt concentration, valency of ions). The biomoleculescan also be coupled to (nano)particles, (micro)beads. The fluorescenceof these modified particles changes depending on whether e.g. ssDNA/RNAor dsDNA/RNA (or other biomolecules) is bound to it. Heating of suchsolutions leads to the same result without the need of specificfluorescent markers bound to the biomolecule. Beside a qualitativediscrimination, also a quantitative thermodynamic analysis is possible.Since the measurement times are in some cases below the relaxation timesof intermolecular reactions, it is not in all cases possible todetermine the thermodynamic parameters like dS (change of entropy), dH(change of enthalpy) and dG (change of Gibbs free energy) directly. Butby using non equilibrium thermodynamics, these can be easily calculated.For the qualitatively discrimination of (i.e.) single nucleotidepolymorphisms, working under non equilibrium condition can increase themeasured differences in stability of the compared molecules. Themeasurement of mismatches in nucleotide sequences is of great importancein medical diagnostics. The method allows identifying hereditarydiseases. It can also be used in pharmaceutical high throughputscreenings for the binding of low molecular weight compounds to nucleicacids. Furthermore the melting of double-stranded (ds) nucleic acidsallows to determine their length.

The measured melting curves of the present invention reproduce theresults measured by established techniques but up to 3000× faster thanPeltier or heating bath based methods, like PCR cycler or fluorimetermethods. The inventive method is much faster since it is not necessaryto heat the volume by direct contact, and the reaction of molecules on aspecific temperature between 0° C. and 100° C. is observed at the sametime. Again, the gist of this embodiment is that temperatures aregenerated and measured with spatial instead of temporal resolution.There are no delays due to heating and cooling times which makes themethod of the present invention very fast. At the same time only thinsheet of liquids are used which decreases the necessary sample volume.In addition the manipulation and analysis of the molecules occurs alloptically without the risk of contamination. This is essential if theanalysis is combined with PCR reactions where any contamination withi.e. human DNA renders an analysis impossible.

By performing the thermal denaturation within e.g. 100 ms, preferably 50ms, it is possible to measure the effect of substances on theDNA/protein stability which are sensitive to high temperatures (e.g. DNAbinding proteins, substances like cyclic-Adenosin Monophosphate (cAMP)).These substances will be damaged or degraded in an experiment withtechniques of the prior art and an effect on the thermal stability isnot detectable with such prior art techniques.

Within the spatial temperature distribution there are temperaturegradients. If the measurement time is longer than the 150 ms (for thehere proposed chamber thickness of e.g. 20 μm) one can measure thethermophoretic movement of the biomolecules (molecules, nanoparticles,microbeads). From this measurements additional information can begathered.

Second Illustrative Embodiment of the Invention

The method according to a second illustrative embodiment (i.e. the aboverecited method relating to measurements wherein the pre-determined timein said second detection of the method of the invention is within arange of 0.5 seconds to 250 seconds, preferably within a range of 0.5 sto 50 s, more preferably within a range of 0.5 s to 40 s) is inparticular useful for a measure of the mobility of molecules in atemperature gradient and its use for biomolecule characterisation. Themethod of the first embodiment described above analyzes molecules in atemperature distribution on a short timescale of milliseconds. Dynamicseffects like thermophoresis can be neglected in this short timeinterval. If molecules are observed for a time period in the order ofseconds, thermophoresis sets in and molecules start moving in thetemperature gradient. This effect drives molecules analogous toelectrophoresis along a gradient to lower temperatures (in some casesthe opposite is also observed). The velocity of the molecules isdirectly proportional to the temperature gradient with a moleculespecific coefficient D_(T) (thermophoretic mobility): v=−D_(T)∇T.

Unexpectedly the thermophoretic mobilities of biopolymers vary stronglywith the chain/molecule length.

The thermophoretic mobility of these molecules varies strongly withmolecule parameters which change the entropy of solvation, size, charge,kind of surface, size of surface, hydrodynamic radius etc. This opensthe possibility to discriminate biomolecules and detect an interactionbetween them (also between nanoparticles/micro beads and biomolecules)(as illustrated in the appended figures, particularly FIG. 4).

Since thermophoresis builds up concentration gradients, the effect iscounteracted by ordinary diffusion. The interplay between these twoeffects leads to a steady state concentration profile which is expressedby the following equation

$\frac{c}{c_{0}} = {{\exp\left\lbrack {{- S_{T}} \times \Delta\; T} \right\rbrack}.}$The concentration at any given point in a temperature distribution issolely dependent on the difference in temperature and not thetemperature gradient any more. The quotient of thermophoretic mobilityD_(T) and ordinary diffusion constant D is called Soret coefficientS_(T) and describes the magnitude of thermophoresis in steady state. Itis exponentially dependent on the temperature difference. Thus theprecision of the measurement is strongly dependent on thereproducibility of the temperature profile.

A typical measurement procedure according to the second embodiment willbe described in the following. In the very beginning an image is takenwithout IR LASER heating to determine the fluorescence intensity of the100% relative concentration level. Then the LASER is turned on. In thisexperimental setup it is possible to focus the laser tightly with a halfwidth below 6 μm to create strong temperature gradients or to use itdefocused (as described above with a temperature profile half width ofe.g. 200 μm). This influences the velocity of the molecules and how fastthe steady state is reached. The necessary temperature increase variesbetween 0.1° C. and 80° C. above ambient temperature (20° C.). If thechamber is e.g. cooled to 0° C., a temperature range between 0.1° C. and100° C. temperature increase can be realized, dependent on the thermalstability of the sample and the magnitude of the thermophoretic effect.In general no high temporal resolution of the image recording isnecessary to determine the thermophoretic effect of the (bio)molecules,(nano)particles, or (micro)beads. The measured signal is the steadystate or close to steady state concentration which is in most casesreached after a few seconds. The signals of most molecules differ strongenough from each other before steady state is reached to identify themwithout any doubt. For the data analysis beside the initialconcentration the concentration at a certain time after LASER heating isturned on is needed (also time courses can be taken). It is sufficientto determine the concentration of a single pixel (i.e. point of maximumtemperature).

Before the laser is turned on there is a homogenous distribution of thebiomolecules. Therefore the fluorescence intensity (or whatever themeasurement signal is), which is direct proportional to theconcentration, has the same magnitude at each point. When the LASER isturned on the concentration distribution changes. The molecules aremoving away from the hot laser focus. Therefore the magnitude of thefluorescence intensity is decreasing until the steady state is reached.This decrease can be measured and thus the characteristics of themolecules can be derived by the theory of thermophoresis and differentmolecules can be discriminated by comparison.

With the methods as disclosed herein, in particular in relation to thesecond embodiment, for means and methods are provided to detect, measureand/or verify a large variety of interactions. For example DNA/DNA,RNA/RNA, protein/protein, protein/DNA, protein/RNA interactions, butalso protein, DNA, RNA interaction with other materials likenanoparticle/microbeads can be measured. The only requirement is alabel, in particular a fluorescent label, bound to one of the molecules.An exception is, e.g. the case of large microbeads, where lightscattering can be used (directly) for detection. If one uses modifiedmicrobeads in a manner that for example DNA single strands can bind tothis modifications, the mobility of this bead is changed due to thebinding. Hence, the method of the second embodiment allows to detectthis binding. Because this modified beads are used in sequencing setupsthe method of the present invention can be applied there. With thepresent method it is possible to detect everything that changes thesize, charge or surface of a molecule. It has been shown that the methodof the present invention is also able to measure specific DNA binding tonanoparticles and polystyrene microbeads via Streptavidin/Biotin (seeFIG. 35). Also interactions between antibody and an epitope aredetectable. Also the binding of a protein to a DNA strand, for exampleof a polymerase, is detectable.

Since no high temporal and spatial resolution is needed the method ofthe second embodiment is cost-efficient and easy to realize. For exampleinstead of a CCD camera an avalanche photodiode can be used (onlyinformation of a single Pixel is needed). If the microfluidic chamber isa capillary (i.e. a microfluidic chamber with high aspect ratio(Length/Width)), no spatial resolution in direction of the width isneeded and for the detection of fluorescence distribution only aline-CCD-camera is needed. This alternative in between CCD-Camera andavalanche photodiode/photomultiplier is very cost efficient and safestime for data evaluation since the integration of fluorescence isperformed by the hardware. The measured systems can exhibitconcentration down to nanomolar without any restriction at higherconcentrations. Also a comparable high degree of contamination istolerated by the methods of the present invention. Measurements are alsopossible in crude extract of cells or in blood. Beside tolerance tocontaminations the method also sustains strong variations in theviscosity of the solution. Measurement can for example be performed inwater or glycerol or in aqueous solution with a gel like consistence.Since measurements are performed in microfluidic chambers the volumeneeded for an experiment is e.g. only 0.5 μl, 1 μl, 2 μl, 5 μl, 10 μl,preferably 2 μl, and can be further reduced. Because of its easycalibration the method has a big advantage compared to FCS (FluorescenceCorrelation Spectroscopy) and can be easily automated. The method isdistinguishable faster than any other method on the market to determineinteractions between biomolecules/nanoparticles/microbeads (i.e.Biacore). The length of short DNA molecules is determined within secondscompared to an hour which is needed by gel electrophoresis, anestablished method in this field. A further advantage is that themeasurements are performed in aqueous solution. It is not necessary tochange the phase in which the molecule is dissolved (gel in gelelectrophoresis or C18, HIC-columns in HPLC). The possibility todifferentiate between single and double stranded DNA that fast opens upnew possibilities in diagnosis as well as scientific research. Oneexample is, e.g. the diagnosis of infectious diseases, like viraldiseases or bacterial infections.

In summary, the inventive method of the second embodiment manipulatesconcentrations of biomolecules/nanoparticles/microbeads in aqueoussolutions by temperature gradients (up to 10 K/μm, in some embodimentsup to 5 K/μm, particularly up to 2 K/μm) established with an IR LASER.In the enclosed example a general theory for thermophoresis in liquidsis described.

The most important features and advantages of the present invention willbe summarized in the following. The method and the device of the presentinvention works all optical, i.e. manipulation and detection is made byoptical means. With the present invention, it is possible to opticallymanipulate molecules down to the size of a single fluorescent dye whichis not possible by optical traps which are limited to sphericalparticles of 500 nm. The method is free of contaminations and easy tominiaturize and to parallelize. That makes it possible to integrate thesystem in established instruments like pipetting robots etc. Heating ofaqueous solutions on the micron scale allows to create temperaturedistributions which renders long heating and cooling periodsunnecessary. The criterions for materials which can be used to buildmeasurement chambers are very unspecific. Another advantage is that IRLASER are getting more and more common in the telecommunication industryand are produced in big quantities. Furthermore the technique offluorescent dye coupling to biomolecules has become a cost-efficientstandard technology. Thus, with the present invention, it is possible tomeasure stability of molecules as well as any kind of interaction (witheach other, the buffer system, other solutes, etc.). Therefore, theinvention is not limited for use in the measurement, detection and/orverification of biomolecules or biological, biomedical, biophysicaland/or pharmacological (in vitro) processes.

With the means and methods of the first embodiment of the presentinvention, melting curves over a large range of temperatures can bemeasured and determined. According to this embodiment, thermophoresisshould be avoided. The temperature achieves a change in the structure,which is detectable via the fluorescence behaviour. The characteristicscan be detected and/or measured over a wide temperate range at one time.None of the known prior art documents discloses an inter- and/orintramolecular reaction induced via temperature. According to the firstembodiment, the laser can be irradiated into the solution via opticalfibres. The light may exit the optical fibres divergent. According to apreferred embodiment of the present invention, the optical system forfocussing the laser can be a single lens.

The means and methods of the second illustrative embodiment of thepresent invention provides the advantages that the effect ofthermophoresis is used in a controlled manner. In particular,concentration changes, induced by thermophoresis effect(s) are measuredvia a change in fluorescence behaviour. Thus, the fluorescence signalrecorded in the second embodiment is primary based on changes ofconcentration and not on changes in the structure of the testedparticles or biomolecules. The second illustrative embodiment includesthat changes in concentration are sensitive to changes in the structureof a particle or molecule. In the known prior art only a maximumtemperature difference of only 2.5 K is disclosed. Moreover, onlymeasurements with a low power laser (320 mW) are described in the priorart. Particularly, a CCD camera can be used for detecting or measuringthe fluorescence of the sample. According to another embodiment, onlyone pixel of the CCD camera has to be used for the measurement or thedetection of the fluorescence light, e.g. only a central 1×1 μm pixelhas to be used. This has the advantage, that further spatial informationmay be neglected according to certain embodiments of the presentinvention.

According to a further aspect of the present invention, temperaturedistributions around the irradiated laser light beam are measured withindependent measurements. These measurements are typically based onknown temperature-dependent fluorescence behaviour of dyes.

Accordingly, as also illustrated in the appended examples, particularembodiments of the present invention relate to the detection ofthermodiffusion or thermophoresis of (bio)molecules or particles, thedetermination of hydrodynamic radii (bio)molecules or particles, thedetection of binding of or between (bio)molecules or particles, thedetection of interactions of or between (bio)molecules or particles, thedetection of conformational changes in (bio)molecules, the detection ofdenaturation of proteins or melting of nucleic acids and to optothermaltrapping of (bio)molecules or particles.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described with reference to the figures in which

FIG. 1 a, 1 b show an fluorescence IR scanning microscope according tothe present invention.

FIG. 2 shows another embodiment of an IR scanning microscope accordingto the invention.

FIG. 3 a-3 d show how melting curves with an radiation of 150 ms can betaken.

FIG. 4 shows a fast SNP detection.

FIG. 5 a-5 b show the mobility in a temperature gradient.

FIG. 6 is an example for a fluorescence dye to be used in the methods ofthe present invention

FIGS. 7-14 show further information with regard to a detailed exampleaccording to the present invention.

FIG. 15: shows the temperature dependence of a fluorescent dye measuredby a fluorimeter with temperature control (Peltier element).

FIGS. 16-24 show particular embodiment of the device according to thepresent invention.

FIG. 25: shows quantification of interaction between biomolecules.

FIG. 26: shows single molecule binding to nanoparticles.

FIG. 27: shows a particular embodiment of the device according to thepresent invention.

FIG. 28: shows the characterization of protein conformation

FIGS. 29-30: show measurements with a sample of fluorescently labelledbovine serum albumin (BSA).

FIG. 31: shows the measurement of the thermo-optical properties of twosamples with/of Green Fluorescent protein (GFP).

FIG. 32: shows a measurement of a particle which is trapped in apotential well created by a spatial temperature distribution

FIG. 33: shows a time series of the thermophoretic motion of silicabeads in a microfluidic chamber.

FIG. 34: shows another example for the “Optothermal Trap”.

FIG. 35: shows the determination of the Soret Coefficient of complexesof nanocrytals (=quantum dot QD) and biomolecules.

FIGS. 36-37: show particular embodiments of the device according to thepresent invention.

FIG. 38: shows an example of a lipid bilayer system.

FIG. 39: shows thermophoresis and thermophoretic trapping of lipidvesicles

DETAILED DESCRIPTION OF DRAWINGS

In the following description of examples of preferred embodiments of theinvention, elements having the comparable technical or physical effecthave the same reference numerals.

FIG. 1 a shows an fluorescence IR scanning microscope according to thepresent invention. The IR scanning microscope is based on a standardfluorescence microscopy setup (e.g. Zeiss AxioTech, Vario). Said devicecomprises: one or more light sources 32, preferably high power LED (e.g.V-Star, Luxeon) to excite the particles. The signal from the particlesmay be collected with an optical system 1, preferably a 40× oilobjective and is separated from the light of the light source by one ormore light separation elements 4, preferably a dichroic mirror. The saidsignal is recorded with one or more detectors 31, preferably CCD Camera(e.g. SensiCam QE, PCO). The beam of an IR LASER 30 (e.g. IPG, Ramanfibre RLD-5-1455) is coupled into microfluidic chamber 45 (e.g. amultiwell plate). The system may comprise other components as wouldordinarily be found in fluorescence and wide field microscopes. Examplesfor means of excitation and detection of fluorescence may be found in:Lakowicz, J. R. Principles of Fluorescence Spectroscopy, KluwerAcademic/Plenum Publishers (1999).

FIG. 1 b shows a further embodiment of an fluorescence IR scanningmicroscope according to the present invention similar to the embodimentsshown in FIG. 1 a. However, the light source 32 is oriented in adifferent way with respect to the dichroic mirror 4. The testing sample50 is sandwiched between two pieces of glass 51, preferably coverslips.

FIG. 2 shows another embodiment of an IR scanning microscope accordingto the invention. According to this embodiment of the present invention,the testing sample 50 is provided in form of a single droplet with themarked particles. The volume (preferably some nanoliter up to somemicroliter) of such a droplet can be easily adjusted such that also thedimensions, i.e. the thickness of the droplet which is irradiated withthe laser beam, is predictable. In this embodiment, the laserbeam, thefluorescently exciting light from the light source 32, preferably a LEDas well as the measured fluorescent light are all focussed by a commonoptical system 1, preferably a microscope objective with high numericalaperture and more preferably a objective with high numerical apertureand high IR transmission. Therefore, the LED, the LASER 30 and thedetector 31, preferably a CCD can be arranged at a common side withrespect to the sample. The exciting light is separated from thefluorescent light by a light separation element 5, preferably a dichroicmirror, which preferably separates different parts of the light spectrumthan the light separation element 4.

FIG. 3 a-d show how melting curves with an radiation of 150 ms can betaken. (a) By measuring the fluorescence of a temperature sensitive dye,the temperature distribution in the microfluidic chamber can bemeasured. (b) shows the radial average of the temperatures measured byfluorescence. (c) Aprox. 150 ms after the IR LASER is turned on an imageof the fluorescently labelled DNA is taken. The high intensity showsmelted ds DNA. From (a) and (c) the melting curve can be determined veryfast (d).

FIG. 4 shows a fast SNP detection. A 16 mer of dsDNA with a singlenucleotide mismatch in the center (blue) is compared with the wild type(black). Within 150 ms both species can be clearly discriminated. Theordinate describes the fraction of dissociated dsDNA. The melting pointis defined by 50% dissociated molecules. It is shifted by 15° C. by asingle mismatch.

FIG. 5 a-5 b show the mobility in a temperature gradient. The figuresshow the change of concentration in the central pixel of a heat spotover time. A few seconds after the start of the measurement the laser isturned on and the concentration decreases until a steady state isreached. The signal allows to distinguish between a 20 mer and 50 merdsDNA (a) as well as between 20 bases ssDNA and 20 basepair dsDNA (b).

FIG. 6 is an example for a fluorescence dye to be used in the methods ofthe present invention (6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein(HEX, SE; C20091) from Invitrogen).

FIGS. 7-14 show further information with regard to a detailed exampleaccording to the present invention. These figures show in particular:

FIG. 7: illustrates how thermodiffusion manipulates the DNAconcentration by small temperature differences within the bulk solution.A thin water film is heated by 2 K along the letters “DNA” with aninfrared laser. For a cooled chamber at 3° C., fluorescently tagged DNAaccumulates to the warm letters. However at room temperature DNA movesinto the cold, showing reduced fluorescence. The chamber is 60 μm thin,containing 50 nM DNA in 1 mM TRIS buffer. Every 50th base pair islabelled with TOTO-1.

FIG. 8 illustrates the salt dependency. (a) Thermodiffusion in water isdominated by ionic shielding and water hydration. (b) Soret coefficientST versus Debye length for carboxyl modified polystyrene beads ofdiameter 1.1, 0.5 and 0.2 μm. Linear plot (left) and logarithmic plot(right). The Soret coefficients are described by equation (2) with aneffective surface charge of σeff=4500 e/μm2 known from electrophoresis.The intercept S_(T)(λ_(DH)=0) is fitted with an hydration entropy perparticle surface of Shyd=−1400 J/(mol Kμm2).

FIG. 9 shows a temperature dependency. (a) The temperature dependence isdominated by the linear change in the hydration entropy Shyd. It shiftsthe salt dependent thermodiffusion ST(λDH) to lower values. The particlesize is 1.1 μm. (b) The Soret coefficient ST increases linearly with thetemperature as expected for a hydration entropy Shyd(T). It depends onthe molecule species, not its size, as seen from the rescaled Soretcoefficients for DNA with different lengths.

FIG. 10 shows a size dependency. (a) For polystyrene beads, the Soretcoefficient scales with the particle surface over four orders ofmagnitude. Measurements are described by equation (2) with an effectivesurface charge density of σeff=4500 e/μm2 and negligible hydrationentropy. The deviation for the bead with 20 nm diameter can beunderstood from an increased effective charge due to the onset of chargenormalization for a ≦λ_(DH). (b) Accordingly, the thermodiffusioncoefficient DT scales linearly with bead diameter. (c) The Soretcoefficient of DNA scales according to S_(T)∝√{square root over (L)}with the length L of the DNA based on equation (2) with an effectivecharge per base pair of 0.12 e. (d) Thermodiffusion coefficient DTdecreases over DNA length with D_(T)∝L^(−0.25), caused by the scaling ofdiffusion coefficient D∝L^(−0.75).

FIG. 11: shows an effective Charge from Thermodiffusion. Effectivecharge is inferred from thermodiffusion using equation (3). Polystyrenebeads (20.2000 nm) (a) and DNA (50-50,000 bp) (b) are measured over alarge size range, impossible with electrophoresis. As expected, theeffective charge of the beads scales with particle surface and linearlywith length of DNA.

FIG. 12: shows the dependency of thermodiffusion from concentration overtime. (a) Diffusion coefficient D was obtained from back diffusion afterswitching off the heat source. (b) D is varied until the finite elementsimulation matches the experiment. (c) Radial depletion of DNA from afocussed 2 K heat spot is monitored over time. (d) Comparison withsimulation with known D yields DT and ST.

FIG. 13 shows a scaling of DNA diffusion coefficients. The diffusioncoefficients as measured in this study at room temperature. The scalingover DNA length matches literature values with two scaling regimes withexponent −1 for short and −0.6 for long DNA33. As approximation,diffusion across the two scaling regimes is well described with anoverall exponent of −0.75.

FIG. 14: shows a simulation of Microfluidic Heating. (a) A 10 μm thinwater film is enclosed between PS walls. Low thermal conduction of thechamber walls allow a thickness independent temperature profile,confirmed by the shown finite element calculation. (b) Convection isslow at maximal velocities of 5 nm/s due to thin chamber and comparablebroad heating focus.

FIG. 15 shows the temperature dependence of a fluorescent dye measuredby a fluorimeter with temperature control (Peltier element).

FIG. 16 illustrates a particular embodiment of a device accordingly tothe present invention. The device may have an substantially arbitraryorientation with respect to the direction of gravitation, preferably thedevice is oriented perpendicular with respect to the direction ofgravitation, more preferably the device is oriented substantiallyparallel or anti-parallel with respect to the direction of gravitation.Preferably the orientation of the device with respect to the testingsample or chamber may be adjusted as shown in FIG. 1 a, FIG. 1 b andFIG. 2. The device comprises: 1: Objective (e.g. 40×, NA 1.3, oilimmersion, ZEISS, “Fluar”); 20: Scanning module, may be Galvano scanningmirror or may be acoustic optic deflector (AOD); 3: cold mirror,preferably high IR-transmission and preferably >90% reflection 350nm-650 nm; 11: beam shaping module to determine laser beam diameter andfocusing, may be a lens system which may comprise one, two or morelenses; 16: Laser fibre coupler w/o collimator; 15: Laser fibre (singlemode or multimode); 30: IR-Laser (e.g. 1455 nm, 1480 nm, 0.1 W-10 W); 4:Dichroic mirror/beam splitter reflecting short wavelength (R>80%),transmitting long wavelength (T>80%); 7: Emission Filter (band pass/longpass); 31: detector, may be a CCD-Camera, Line-Camera, PhotomultiplierTube (PMT), Avalanche Photodiode (APD), CMOS-Camera; 6: ExcitationFilter (e.g. band pass/long pass); 10: lens system to determine the beamproperties of the excitation light source, may comprise one, two or morelenses; 32: Excitation light source, may be Laser, Fibre Laser,Diode-Laser, LED, HXP, Halogen, LED-Array, HBO. Preferably the listedparts are enclosed in a housing; a cold mirror is a specializeddielectric mirror, a dichromatic interference filter that operates overa very wide temperature range to reflect the entire visible lightspectrum while very efficiently transmitting infrared wavelengths.

FIG. 17 shows an embodiment of the invention according to FIG. 16,wherein the scanning module 20 is replaced by a fix mirror 21,preferably a silver mirror.

FIG. 18 shows an embodiment of the invention according to FIG. 16,wherein the scanning module 20 is replaced by a fix mirror 21,preferably a silver mirror, and wherein a shutter 33 for controlling theIR LASER radiation is added and wherein a line forming module 12, may bea cylinder lens system or preferably a Powell lens, are added.

FIG. 19 shows a further embodiment of the device according to thepresent invention, in particular a confocal setup. The device may havean arbitrary orientation with respect to the direction of gravitation,preferably the device is oriented substantially perpendicular withrespect to the direction of gravitation, more preferably the device isoriented substantially parallel or anti-parallel with respect to thedirection of gravitation. Preferably the orientation of the device withrespect to the testing sample or chamber may be adjusted as shown inFIG. 1 a, FIG. 1 b and FIG. 2. The device comprises: 1: Objective (e.g.40×, NA 1.3, oil immersion, ZEISS “Fluar”); 2: hot mirror, highIR-reflection, visible light transmission>80%; 4: Dichroic mirrorreflecting short wavelength (R>80%), transmitting long wavelength(T>80%); 7: Emission Filter (band pass/long pass); 31: Detector, may bePhotomultiplier Tube (PMT), Avalanche Photodiode (APD); 13: Pinholeaperture; 10: lens system to determine the beam properties of theexcitation light source, may comprise one, two or more lenses; 32:excitation light source preferably a laser, more preferably a fibrecoupled laser 11: beam shaping module to determine laser beam diameterand focusing, preferably lens system which may comprise one, two or morelenses; 16: Laser fibre coupler w/o collimator; 15: Laser fibre (singlemode or multimode); 30: IR-Laser (e.g. 1455 nm, 1480 nm, 0.1 W-10 W);33: Shutter; 14: pinhole aperture in confocale position to laser pinhole13 or to laser fibre coupler 17 whereas the pinhole 13 may not beneeded, preferably if a fibre coupled laser is used as excitation lightsource 32; 18 Laser fibre may be single mode or may be multi mode.

FIG. 20 shows a further embodiment of the device according to thepresent invention. The device may have an arbitrary orientation withrespect to the direction of gravitation, preferably the device isoriented substantially perpendicular with respect to the direction ofgravitation, more preferably the device is oriented substantiallyparallel or anti-parallel with respect to the direction of gravitation.Preferably the orientation of the device with respect to the testingsample or chamber may be adjusted as shown in FIG. 1 a, FIG. 1 b andFIG. 2. The device comprises: 1: Objective (e.g. 40×, NA 1.3, oilimmersion, ZEISS “Fluar”); 2: hot mirror, high IR-reflection, visiblelight transmission>80%; 4: Dichroic mirror reflecting short wavelength(R>80%), transmitting long wavelength (T>80%); 6: Excitation Filter(e.g. band pass/long pass); 7: Emission Filter (band pass/long pass);10: lens system to determine the beam properties of the excitation lightsource; 31: Detector, may be Photomultiplier Tube (PMT), AvalanchePhotodiode (APD); 32: excitation light source 16: Laser fibre couplerw/o collimator; 15: Laser fibre (single mode or multimode); 30: IR-Laser(e.g. 1455 nm, 1480 nm, 0.1 W-10 W); a hot mirror is a specializeddielectric mirror, a dichromatic interference filter often employed toprotect optical systems by reflecting heat back into the light source.Hot mirrors can be designed to be inserted into the optical system at anincidence angle varying between zero and 45 degrees, and are useful in avariety of applications where heat build-up can damage components oradversely affect spectral characteristics of the illumination source. Bytransmitting visible light wavelengths while reflecting infrared, hotmirrors can also serve as dichromatic beam splitters for specializedapplications in fluorescence microscopy.

FIG. 21 shows an embodiment of the invention according to FIG. 20,wherein a shutter 33 is added to control the IR laser radiation.

FIG. 22 shows an embodiment of the invention according to FIG. 20,wherein a shutter 33 and a line forming module 12, preferably a lenssystem comprising one, two or more lenses or more preferably a Powelllens are added to control the IR laser radiation.

FIG. 23 shows an embodiment of the invention according to FIG. 16,wherein the scanning module 20 is replaced by a fix mirror 21,preferably a silver mirror and wherein an additional light separationelement 5, preferably a dichroic mirror, which preferably separatesdifferent parts of the light spectrum than the light separation element4 is added and wherein an emission filter 8 is added, preferablytransmitting another range of wavelength than the emission filter 7 andwherein a second detector 31 is added which detects the signal passingthrough the filter 8.

FIG. 24 shows a further embodiment of the device; 31: CCD camera; 7:Emission filter (e.g. bandpass or longpass); 4: light separationelement, preferably a dichroic minor for splitting excitation lightpathway and emission light pathway; 1: Objective; 45: Chamber; 10: Lenssystem for focusing IR-Laser onto sample; 20: scanning module, may begalvanometric mirrors or an acoustic optical deflector; 16: Laser fibrecoupler with collimating optics; 15: Laser fibre; 30: IR-Laser; 6:Excitation Filter; 10: optical system for excitation, may be a lenssystem comprising one, two or more lenses; 32: Light source (HXP,LED);46: xyz-Translation stage for laser positioning, may be automated,preferably may be automated for scanning the chamber. 47: optical systemfor beam shaping of the IR Laser, may be a lens system comprising one,two or more lenses or may be a objective, preferably with high IRtransmission.

FIG. 25 shows a quantification of interaction between biomolecules. 100nM of a fluorescently labelled antibody (anti-Interleukin 4) aretitrated with various amounts interleukin. (left) The spatialfluorescence distribution in steady state is measured. Three curves with5 nM, 80 nM and 300 nM are shown exemplarily. The signal changesdramatically from fluorescence decrease to a fluorescence increase.Integration of the fluorescence profile up to 80 μm (distance from theheated centre) allows to determine the number of complexes in solution.(right) The concentration of free Interleukin 4 can be calculatedplotted versus the concentration formed complex. These data can befitted to determine the K_(D).

FIG. 26 shows a single molecule binding to nanoparticles. The Soretcoefficient of nanocrystals in complex with PEG molecules is measured byevaluating the concentration profile in steady state. The Soretcoefficient increases linearly with the number of PEG moleculescovalently bound to the particle. PEG molecules with a higher molecularweight show a steeper increase in the Soret coefficient. The PEGmolecules shown here are comparable in size to proteins or short DNAmolecules, which can be detected in the same manner.

FIG. 27 shows a further embodiment of a device according to the presentinvention. The receiving means for receiving the sample probe 50 is acapillary 40 with inner diameter 5 μm to 500 μm such that the thicknessof the sample probe is small in the direction perpendicular to the laserbeam. The first valve 41 and the second valve 42 are provided for thecontrolled input/output of the sample probe 50 in/from the capillary 40.The capillary is mounted on a solid support 43, preferably a materialwith good thermal conductivity, e.g. aluminium, copper. ThePeltier-element 44 is mounted on the solid support 43 such that thecapillary 40 can be cooled.

FIG. 28 shows the characterization of protein conformation.Thermo-optical characterization provides the means to characterize theconformation of a protein in solution. The temperature of a solutioncontaining Bovine Serum Albumin (BSA) is cooled down to 0° C. Startingfrom this temperature the Soret coefficient is measured at differenttemperatures which are increased in a stepwise manner up to 60° C. TheSoret coefficient is negative, up to values close to thermaldenaturation, where a sudden jump to positive Soret coefficients isobserved. At physiological temperatures (30-40° C.) the Soretcoefficient does not change much. In this temperature range the proteinhas to have similar properties to perform its tasks. Because of thetight relation between structure and function, the conformation ispreserved in this temperature range. The results are confirmed by theexperiment shown on the right, which starts at high temperatures. TheSoret Coefficients are still positive below 50° C. since themeasurements are faster than the time the protein needs for refolding.After a certain time span (i.e. 20 minutes) the values reach thenegative Soret coefficients obtained in the measurements started at lowtemperatures. A following temperature increase reproduces the negativeSoret coefficient measured in the experiment starting at lowtemperatures.

FIG. 29 shows measurements with a sample of fluorescently labelledbovine serum albumin (BSA). A sample of fluorescently labelled bovineserum albumin (BSA) has been split in two parts. One is only exposed toambient temperatures, while the other half is heated up to 100° C. forseveral minutes. The thermo-optical properties of both samples (nativeand denatured) are measured at different power of the infrared laser(i.e. maximal temperature increase of 5° or 10° C.). As can be seen fromthe figure, the fluorescence of the denatured protein is lower than thefluorescence of the native protein. This is explained as follows. Thefluorescence dye of both samples shows the same decrease in fluorescencedue to the increase in temperature (i.e. temperature sensitivity of thefluorescence). But the denatured protein shows a positive thermophoreticmobility (i.e. moves to the cold), while the native protein has anegative thermophoretic mobility (i.e. moves to the hot). Theaccumulation at elevated temperatures is the reason, why the decrease influorescence is lower for the native protein, while the denaturedprotein is, in addition to temperature dependency, depleted from theregion of elevated temperature. The differences between both samples isfurther increases by raising the temperature (i.e. maximum temperatureof 10° C.), since positive and negative thermophoresis is enhanced.

FIG. 30 measurements with a sample of fluorescently labelled bovineserum albumin (BSA). A sample of fluorescently labelled bovine serumalbumin (BSA) has been split in two parts. One is only exposed toambient temperatures, while the other half is heated up to 100° C. forseveral minutes (i.e. irreversibly denatured). The thermo-opticalproperties of both samples (native and denatured) are measured at 800 mApower of the infrared laser (i.e. maximal temperature increase of 20°C.). As can be seen from the figure, the fluorescence of the denaturedprotein is lower than the fluorescence of the native protein. This isexplained as follows. The fluorescence dye of both samples shows thesame decrease in fluorescence due to the increase in temperature (i.e.temperature sensitivity of the fluorescence). But the denatured proteinshows a positive thermophoretic mobility (i.e. moves to the cold), whilethe native protein has a negative thermophoretic mobility (i.e. moves tothe hot). The accumulation at elevated temperatures is the reason, whythe decrease in fluorescence is lower for the native protein, while thedenatured protein is, in addition to temperature dependency, depletedfrom the region of elevated temperature. Interestingly, by approachingthe denaturing temperature (i.e. 50° C.) of the protein the amplitudesof the native and denatured protein approach each other an areessentially the same. This means that by measuring the amplitude of thefluorescence change an comparison to the reference sample allows todetect the melting temperature of a protein and to discriminate betweennative and denatured form of a protein. And to detect a shift in meltingtemperature due to interactions of the protein with other biomoleculesor small molecules (e.g. drug candidates).

FIG. 31 shows the measurement of the thermo-optical properties of twosamples with/of Green Fluorescent protein (GFP). The thermo-opticalproperties of two samples of Green Fluorescent protein (GFP) aremeasured. In the first sample only GFP is present, while in the secondsample the GFP is mixed with a 2 fold excess of an antibody fragment,specifically binding to GFP. In both cases, first the fluorescence isrecorded without laser heating. Then the fluorescence excitation isturned off and the IR-laser radiation is turned on. The laser is turnedoff after a few seconds of heating and the fluorescence excitation isturned on at the same time. The relaxation of the spatial fluorescencedistribution (i.e. concentration distribution) to a homogeneous state isrecorded for a few seconds. As can be observed from the figure, in thesample with the two interacting species (i.e. GFP and the antibodyfragment) the fluorescence profile needs more time to relax. This isexplained by slower diffusion of the larger complex. The time evolutionof the fluorescence profile is analyzed via a software tool to determinethe diffusion constant. By using the Stokes-Einstein relation, anhydrodynamic radius is attributed to the diffusion constant. In case ofthe free GFP this is 5 nm and the complex has an radius of 10 nm.

FIG. 32 shows a measurement of a particle which is trapped in apotential well created by a spatial temperature distribution. (a) Aparticle is trapped in a potential well created by a spatial temperaturedistribution. For silica particles the well is deepest at hightemperatures. The fluctuations are recorded via a CCD camera (at t=1 s,2 s, 3 s, 4 s, 5 s, 6 s, 7 s) and (b) the positions are tracked bySoftware with nanometer resolution. (c) A histogram is calculated fromthe positional information. The width of the distribution is verysensitive to the thermo-optical properties of the particle. If moleculesbind to the surface of the particle, the effective potential for thebead changes and the amplitude of the fluctuations increases ordecreases. By observing the amplitude change over time, a kineticbinding curve can be measured.

FIG. 33 shows a time series of the thermophoretic motion of silica beadsin a microfluidic chamber. Time series of the thermophoretic motion ofsilica beads in a microfluidic chamber. In the beginning (image 1),without laser heating, the beads are almost equally distributed. Theblack circle shows the position of the laser focus. The following imagesshow the development of the particle distribution in the next threeseconds after the heating laser is turned on. The particles areattracted by the heat source and accumulate at the point of highesttemperature. The accumulation if observed because these particles have anegative thermophoretic mobility. A Particle with positivethermophoretic mobility can be trapped by heating e.g. a circle aroundit.

FIG. 34 shows another example for the “Optothermal Trap”. AnotherExample for the “Optothermal Trap”: Several 1 μm beads are trapped atthe bright spot at the centre of the image. The chamber is moved whereasthe laser focus was kept fixed. The image is an addition of about 30single images. As one can see, all beads were moved with the chamber, sothe addition of the single images results in lines for the singleparticles. The trapped beads were hold on one position. No movement ofthe trapped beads was detected. The halo and the high intensity of thetrapped beads is the result of the addition of the single images.

FIG. 35 shows the determination of the Soret Coefficient of complexes ofnanocrytals (=quantum dot QD) and biomolecules. The Soret Coefficient ofcomplexes of nanocrytals (=quantum dot QD) and biomolecules isdetermined by relating the spatial concentration distribution to aspatial temperature distribution. Three different samples have beenanalyzed. First a nanocrystal without protein modification is measured(QD), followed by a sample nanocrystals modified with the proteinstreptavidin (QD+Strep.) (approximately 5 proteins per nanocrystal). Bybinding the protein to the nanocrystal, the Soret-Coefficient isstrongly increased. By adding a single stranded DNA to the sample (oneDNA per Particle), the Soret coefficient is increased further(QD+Strep.+DNA).

FIG. 36 shows a further embodiment of the invention according to FIG.20, wherein a stage 43 carrying a temperature control element 44 and thechamber 45 is connected to the optical system via connectors 48. Theoptical system 1 may also be also comprise a TIRF (total internalreflection fluorescence) objective so that Thermophoresis can bemeasured in direction of the Laser beam.

FIG. 37 shows a further embodiment of the invention according to FIG.20, wherein a shutter 33 and a line forming module 12, preferably a lenssystem comprising one, two or more lenses or more preferably a Powelllens are added to control the IR laser radiation and wherein theemission filter 7 is replaced by an optical instrument 22 which may be aspectrograph, polychromator or monochromator or combinations of one ormore of these, e.g. an optical instrument which transforms differentintervals of wavelength/frequency of light into different intervals ofangles/distances or different places for example on a CCD.

FIG. 38 shows an example for a lipid bilayer model system. A fraction ofthe layer constituting lipids is coupled to a surface (e.g. via asulfhydryl-peptide to a gold surface) Transmembrane proteins or membraneassociated proteins are inserted into the lipid bilayer. In additionalso soluble proteins may be present in the aqueous solution on top ofthe membrane. By infrared laser absorption of the aqueous solution atemperature gradient can be established within the membrane. This waythe thermo-optical properties like stability, interaction andconformation may can be measured for a fluorescently labelled compound(i.e. lipid, membrane protein or soluble protein).

FIG. 39 shows thermophoresis and thermophoretic trapping of lipidvesicles. The images (200×200 μm) show a solution of unilamelarvesicles, without (a) and after 10 seconds of infrared laser heating.(a) shows a uniform distribution of the vesicles. The infrared laserheats the solution locally to a maximum temperature of 15° C. above roomtemperature of 20° C. As (b) shows the local temperature increaseattracts the vesicles (i.e. negative thermophoresis) and confines theirposition to a region close to the center of the heat spot. The regionaround the heat spot is depleted of vesicles. Vesicles closer to theedge of the field of view experience only a small gradient an are notattracted within the time span of 10 seconds. Broadening of thetemperature profile would also attracted these particles much faster.

EXAMPLES

The following detailed example illustrates the invention without beinglimiting.

Example 1 Thermodiffusion

Molecules drift along temperature gradients, an effect calledthermophoresis, Soret-effect or thermodiffusion. In liquids, itstheoretical foundation is subject of a long standing debate. Using a newall-optical microfluidic fluorescence method, we present experimentalresults for DNA and polystyrene beads over a large range of particlesize, salt concentration and temperature. The data supports a unifyingtheory based on the solvation entropy. Stated in simple terms, the Soretcoefficient is given by the negative solvation entropy, divided by kT.The theory predicts the thermodiffusion of polystyrene beads and DNAwithout any free parameters. We assume a local thermodynamic equilibriumof the solvent molecules around the molecule. This assumption isfulfilled for moderate temperature gradients below the fluctuationcriterion. Above this criterion, thermodiffusion becomes non-linear. Forboth DNA and polystyrene beads, thermophoretic motion changes sign atlower temperatures. This thermophilicity towards lower temperatures isattributed to an increasing positive entropy of hydration, whereas thegenerally dominating thermophobicity is explained by the negativeentropy of ionic shielding. The understanding of thermodiffusion setsthe stage for detailed probing of solvation properties of colloids andbiomolecules. For example, we successfully determine the effectivecharge of DNA and beads over a size range which is not accessible withelectrophoresis.

Introduction.

Thermodiffusion has been known for a long time, but its theoreticalexplanation for molecules in liquids is still under debate. The searchfor the theoretical understanding is motivated by the fact thatthermodiffusion in water might lead to powerful all-optical screeningmethods for biomolecules and colloids. Equally well, thermodiffusionhandles and moves molecules all-optically and therefore can complementwell established methods as for example electrophoresis oroptical-tweezers. For the latter, forces of optical tweezers scale withparticle volume and limit this method to particles only larger than 500nm. Electrophoresis does not suffer from force limitations, but isdifficult to miniaturize due to electrochemical reactions at theelectrodes.

On the other hand, thermodiffusion allows the microscale manipulation ofeven small particles and molecules. For example, 1000 bp DNA can bepatterned arbitrarily in bulk water (FIG. 7). The temperature pattern“DNA”, heated by 2 K, was written into a water film with an infraredlaser scanning microscope. The concentration of 1000 bp DNA was imagedusing a fluorescent DNA tag. In an overall cooled chamber at 3° C., DNAaccumulates towards the heated letters “DNA” (negative Soret effect)whereas at room temperature DNA is thermophobic (positive Soret effect)as seen by the dark letters.

In the past, the apparent complexity of thermodiffusion prevented a fulltheoretical description. As seen for DNA in FIG. 13, moleculescharacteristically deplete from regions with an increased temperature,but they can also show the inverted effect and accumulate³. Moreover,the size scaling of thermodiffusion recorded by thermal field flowfractionation (ThFFF) showed fractional power laws with a variety ofexponents which are hard to interpret⁴. The latter effect was resolvedrecently by revealing nonlinear thermophoretic drift for the strongthermal gradients used in ThFFF.

A variety of methods were used to measure thermodiffusion, mostly in thenonaqueous regime. They range from beam deflection^(3,7), holographicscattering^(8,9), electrical heating to thermal lensing. Recently wehave developed a fluorescence microfluidic imaging technique^(13,14)which allows the measurement of thermodiffusion over a wide moleculesize range without artifacts induced by thermal convection. Highlydiluted suspensions can be measured and therefore particle-particleinteractions do not have an influence. We only apply moderatetemperature gradients. In the following we used this method to confirm astraightforward theoretical explanation of thermodiffusion.

Theoretical Approach.

For diluted concentrations, it is generally assumed that thethermodiffusive drift velocity {right arrow over (v)} depends linearlyon the temperature gradient ∇T with a proportionality constant whichequals the thermodiffusion coefficient D_(T): {right arrow over(v)}=−D_(T)∇T. In steady state, thermodiffusion is balanced by ordinarydiffusion. Constant diffusion and thermodiffusion coefficients both leadto an exponential depletion law¹⁶ c/c₀=exp[−(D_(T)/D)(T−T₀)], with theconcentration c depending on the temperature difference T−T₀ only. Theconcentration c is normalized by the boundary condition of theconcentration c_(o) with temperature T₀. The Soret coefficient isdefined as ratio S_(T)=D_(T)/D which determines the magnitude ofthermodiffusion in the steady state. While the above exponentialdistribution could motivate an approach based on Boltzmann equilibriumstatistics, it is commonly argued that thermodiffusion without exceptionis a local non-equilibrium effect that requires fluid dynamics, forcefields or particle-solvent potentials¹⁷⁻²⁰. However, in two previouspapers¹⁶ we demonstrated that for moderate temperature gradients, thethermal fluctuations of the particle are the basis for a localequilibrium. This allows the description of the thermodiffusive steadystate by a succession of local Boltzmann laws, yieldingc/c₀=exp[(G(T₀)−G(T))/kT] with G the Gibbs-free enthalpy of the singleparticle-solvent system. Such an approach is only valid if thetemperature gradient ∇T is below a threshold ∇T<(aS_(T))⁻¹ which isgiven by the particle fluctuations with the hydrodynamic radius a andSoret coefficient S_(T), as shown recently. For larger temperaturegradients, thermodiffusive drift is nonlinearly dependent on thetemperature gradient. In the present study, temperature gradients belowthis limit were used so that thermodiffusion is measured at localthermodynamic equilibrium conditions.

Local thermodynamic equilibrium allows the derivation of a thermodynamicfoundation of the Soret coefficient. The local Boltzmann distributionrelates small concentration changes δc with small Gibbs-Free Energydifferences: δc/c=−δG/kT. We equate this relation with a locally,linearized thermodiffusion steady state given by δc/c=−S_(T)δT and thusfind the Soret coefficient by the temperature derivative of GS _(T) =D _(T) /D=(kT)⁻¹ ×∂G/∂T  (1)

Whereas above relation is sufficient for the following derivation, itcan be generalized by locally applying the thermodynamic relationdG=−SdT+Vdp+μdN. For single particles at a constant pressure we findthat the Soret coefficient equals the negative entropy of theparticle-solvent system S according to S_(T)=−ΔS/kT. This relation isnot surprising since the entropy is by definition related with thetemperature derivative of the free enthalpy.

The above general energetic treatment is inherent in previouslydescribed approaches based on local equilibrium²², including thesuccessful interpretation of thermoelectric voltages of dilutedelectrolytes^(24,25) which are described by energies of transfer.Recently, the non-equilibrium approach by Ruckenstein was applied tocolloids²⁷ with the characteristic length 1 assigned to the Debye lengthλ_(DH). If instead it would assigned the characteristic length accordingto 1=2a/3 with the particle radius a, the Ruckenstein approach wouldactually confirm the above local equilibrium relation (1) for the Soretcoefficient. Measurements on SDS micelles²⁷ appeared to confirm thisnon-equilibrium approach, but for the chosen particles the competingparameter choices 1=2a/3 and 1=λ_(DH) yielded comparable values. Thusthe experiments could not distinguish between the competing theories.

Here, we will use the above local equilibrium relations to derive theSoret coefficient for particles larger than the Debye length in aqueoussolutions and put the results to rigorous experimental tests. Twocontributions dominate the particle entropy S in water (FIG. 8 a): theentropy of ionic shielding and the temperature sensitive entropy ofwater hydration. The contribution from the entropy of ionic shielding iscalculated with the temperature derivative of the Gibbs-freeenthalpy^(27,28) G_(ionic)=Q_(eff) ²λ_(DH)/[2A∈∈₀] with the effectivecharge Q_(eff) and particle surface A. Alternatively, this enthalpy canbe interpreted as an electrical field energy G_(ionic)=Q_(eff) ²/[2C] inthe ionic shielding capacitor C. We neglect the particle-particleinteractions since the fluorescence approach allows the measurement ofhighly diluted systems. To obtain the Soret coefficient, temperaturederivatives consider the Debye length λ_(DH)(T)=√{square root over(∈(T)∈₀kT/(2e²c_(S)))}{square root over (∈(T)∈₀kT/(2e²c_(S)))} and thedielectric constant ∈(T). Both temperature derivatives give rise to afactor β=1−(T/∈)∂∈/∂∈. The effective charge Q_(eff) is largelytemperature insensitive which was confirmed by electrophoresisindependently²⁹. Such a dependence would be unexpected as the stronglyadsorbed ions dominate the value of the effective charge.Experimentally, we deal with colloids exhibiting flat surfaces, i.e. theparticle radius is larger than λ_(DH). In this case chargerenormalization does not play a role and we can introduce an effectivesurface charge density σ_(eff)=Q_(eff)/A per molecule area A. From thetemperature derivation according to equation (1), the ionic contributionto the Soret coefficient is S_(T) ^((ionic))=(Aβσ_(eff)²λ_(DH))/(4∈∈₀kT²). A similar relation was derived for charged micellesrecently²³, however without considering the temperature dependence ofthe dielectric coefficient ∈. Next, the contribution to the Soretcoefficient from the hydration entropy of water can be directly inferredfrom the particle area specific hydration entropy S_(hyd)=S_(hyd)/A,namely S_(T) ^((hyd))=−As_(hyd)(T)/kT. Finally, the contribution fromthe Brownian motion is derived as S_(T)=1/T by inserting the kineticenergy of the particle G=kT into equation (1). However this contributionis very small (S_(T)=0.0034/K) and can be neglected for the moleculesunder consideration. The contributions from ionic shielding andhydration entropy add up to:

$\begin{matrix}{S_{T} = {\frac{A}{kT}\left( {{- s_{hyd}} + {\frac{\beta\;\sigma_{eff}^{2}}{4{ɛɛ}_{0}T} \times \lambda_{DH}}} \right)}} & (2)\end{matrix}$

The Soret coefficient S_(T) scales linearly with particle surface A andDebye length λ_(DH). We test equation (2) by measuring S_(T) versus thesalt concentration, temperature and molecule size. In all casesthermodiffusion is quantitatively predicted without any free parameters.We used fluorescence single particle tracking to follow carboxylmodified polystyrene (PS) beads (Molecular Probes F-8888) of 1.1 μm and0.5 μm diameter at 25 attomolar concentration, dialyzed into 0.5 mMTris-HCl at pH 7.6. Thermodiffusion of particles ≦0.2 μm is measured bythe fluorescence decrease that reflects the bulk depletion of theparticles¹³. The chamber thickness of 20 μm damped the thermalconvection to negligible speeds¹⁶. The experimental design also excludesthermal lensing and optical trapping¹⁶. Debye lengths λ_(DH) weretitrated with KCl (see supplementary materials).

Salt Dependence.

FIG. 8 b shows the Soret coefficients of polystyrene beads withdifferent sizes versus λ_(DH). The Soret coefficients scale linearlywith a small intercept at λ_(DH)=0 and confirm the λ_(DH)-dependence ofequation (2). For smaller diameters of the beads the Soret coefficientsscale with the particle surface area A (FIG. 8) as expected fromequation (2). To check whether equation (2) also quantitatively explainsthe measured Soret coefficients, we inferred the effective charge of thebeads by electrophoresis (see supplementary material). Using 40 nm beadswith identical carboxyl surface modifications at λ_(DH)=9.6 nm, wefluorescently observed free-flow electrophoresis and corrected forelectroosmosis, finding an effective surface charge density ofσ_(eff)=4500±2000e/μm². This value is virtually independent from theused salt concentrations²⁹. Using this inferred effective charge,equation (2) fits the Soret coefficient for various bead sizes and saltconcentrations well (FIG. 8 b, solid lines).

The intercept S_(T)(λ_(DH)=0), where ionic contributions are zero, alsoscales with particle surface and is described by a hydration entropy perparticle surface of S_(hyd)=−1400 J/(mol Kμm²). The value matches theliterature values for similar surfaces reasonably well^(30,31). Forexample, Dansyl-Alanine, a molecule with surface groups comparable withpolystyrene beads, was measured to have a hydration entropy³⁰ of −0.13J/(mol K) at a comparable temperature. Linear scaling with its surfacearea by using a radius of a=2 nm results in a value of S_(hyd)=−2500J/(mol Kμm²), in qualitative agreement with our result. The hydrationentropy is a highly informative molecule parameter which is notoriouslydifficult to measure, yielding an interesting application forthermodiffusion.

Temperature Dependence.

Hydration entropies S_(hyd) in water are known to increase linearly withdecreasing temperatures³⁰⁻²². Since the slope of the ionic contributionof S_(T) versus λ_(DH) is with high precision temperature insensitivefor water (β(T)/(∈T²)≅const), only the intercept is expected to decreaseas the overall temperature of the chamber is reduced. This is indeed thecase, as seen from the temperature dependence of beads with 1.1 μmdiameter (FIG. 9 a, T=6 . . . 29° C.). We infer from the interceptS_(T)(λ_(DH)=0) that the hydration entropy changes sign at about 20° C.As seen for DNA in FIG. 7, hydration entropy can dominatethermodiffusion at low temperatures and move molecules to the hot(D_(T)<0).

The properties of hydration entropy lead to a linear increase of S_(T)over temperatures at fixed salt concentration as measured for 1.1 μmbeads and DNA (FIG. 9 b). We normalize S_(T) by dividing with S_(T)(30°C.) to compensate for molecule surface area. The slopes of S_(T) overtemperature differ between beads and DNA. However the slope does notdiffer between DNA of different size (50 base pairs versus 10000 basepairs). Based on equation (2), this is to be expected since thetemperature dependence of the hydration entropy only depends on the typeof surface of the molecule, not its size. We measured the diffusioncoefficients of the DNA species at the respective temperatureindependently. Within experimental error, changes in the diffusioncoefficient D match with the change of the water viscosity without theneed to assume conformational changes of DNA over the temperature range.Please note that the change of the sign of the DNA Soret coefficient issituated near the point of maximal water density only by chance. There,the two entropic contributions balance. For polystyrene beads atλ_(DH)=2 nm for example, the sign change is observed at 15° C. (FIG. 9a). An increased Soret coefficient over temperature was reported foraqueous solutions before³, however with a distinct nonlinearity which weattribute to remnant particle-particle interactions.

Size Dependence of the Beads.

The Soret coefficient was measured for carboxyl modified polystyrenebeads in diameter ranging from 20 nm to 2 μm (Molecular Probes, F-8888,F-8795, F-8823, F-8827). Beads of diameter 0.2 μm, 0.1 μm, 0.04 μm and0.02 μm were diluted to concentrations of 10 pM, 15 pM, 250 pM and 2 nM,and its bulk fluorescence was imaged over time to derive D_(T) andD^(13,16) from the depletion and subsequent back-diffusion. Larger beadswith a diameter of 1.9 μm, 1.1 μm and 0.5 μm were diluted toconcentrations of 3.3 aM, 25 aM and 0.2 pM, and measured with singleparticle tracking⁶. The solutions were buffered in 1 mM Tris pH 7.6 withλ_(DH)=9.6 nm. In all cases interactions between particles can beexcluded. Care was taken to keep the temperature gradient in the localequilibrium regime.

We find that the Soret coefficient scales with particle surface overfour orders of magnitude (FIG. 10 a). The data is described well withequation (2) with an effective surface charge density of σ_(eff)=4500e/μm² and neglected hydration entropy contribution. The 5-fold too lowprediction for the smallest particle (20 nm diameter) can be explainedby charge renormalization since its radius is smaller than λ_(DH).

The diffusion coefficient D for spheres is given by the Einsteinrelation and scales inversely with radius D∝1/a. Inserting equation (2)into S_(T)=D_(T)/D, the thermodiffusion coefficient D_(T) is expected toscale with particle radius a. This is experimentally confirmed over twoorders of magnitude (FIG. 10 b). These findings of ours contradictseveral theoretical studies claiming that D_(T) should be independent ofparticle size^(17-20,27), based on ambiguous experimental results fromthermal field flow fractionation (ThFFF)⁴ which were probably biased bynonlinear thermodiffusion in large thermal gradients.

Size Dependence of DNA.

Whereas polystyrene beads share a very narrow size distribution as acommon feature with DNA molecules, beads are a much less complicatedmodel system. Beads are rigid spheres which interact with the solventonly at its surface. In addition, the charges reside on the surfacewhere the screening takes place. Thus the finding that thermodiffusionof flexible and homogeneously charged DNA is described equally welldescribed with equation (2) is not readily expected and quiteinteresting (FIG. 10 c,d).

We measured DNA sizes with 50 base pairs to 48502 base pairs in 1 mMTRIS buffer (λ_(DH)=9.6 nm) at low molecule concentrations between 1 μM(50 bp) and 1 nM (48502 bp). Only every 50th base pair was stained withthe TOTO-1 fluorescent dye. The diffusion coefficient was measured byback-diffusion after the laser was turned off and depends on the lengthL of the DNA in a non-trivial way. The data is well fitted with ahydrodynamic radius scaling a ∝L^(0.75). This scaling represents aneffective average over two DNA length regimes. For DNA molecules longerthan approximately 1000 bp, a scaling of 0.6 is found³³ whereas shorterDNA scales with an exponent of ≅1 (see supplementary material).

We can describe the measured Soret coefficient over three orders ofmagnitude of DNA lengths with equation (2) if we assume that effectivecharge of the DNA is shielded at the surface of a sphere with thehydrodynamic radius a. Due to the low salt concentration (λ_(DH)=9.6nm), such globular shielding is reasonable. Not only is theexperimentally observed scaling of the Soret coefficient with the squareroot of its length correctly predicted based on equation (2)(S_(T)∝Q_(eff) ²/a²∝L²/L^(1.5)∝L^(0.5)), also the Soret coefficient isfully described in a quantitative manner (FIG. 10 c, solid line), withan effective charge of 0.12 e per base, matching well with literaturevalues³⁴ ranging from 0.05 e/bp to 0.3 e/bp.

As shown in FIG. 10 d, the thermodiffusion coefficient for DNA dropswith DNA length according to D_(T)=DS_(T)∝Q_(eff)²/a³∝L²/L^(2.25)∝L^(−0.25). Thus, shorter DNA actually drifts faster ina temperature gradient than longer DNA. It is important to point outthat this finding is in no contradiction to experimental findings of aconstant D_(T) over polymer length in non-aqueous settings⁹. Accordingto equation (1), the thermodynamic relevant parameter is the Soretcoefficient, determined by the solvation energetics. The argument²⁰ thatpolymers have to decouple into monomers to show a constant D_(T) merelybecomes the special case where the solvation energetics determine bothS_(T) and D with equal but inverted size scaling. In accordance with ourlocal energetic equilibrium argument, S_(T) and not D_(T) dominatesthermodiffusion also for non-aqueous polymers near a glass transition³⁵.Here, S_(T) is constant whereas D_(T) and D scale according to anincreased friction. However for a system of DNA in solution, where longranging shielding couples the monomers, a constant D_(T) over polymerlength cannot be assumed a priori (FIG. 10 d).

Effective Charge.

The effective charge Q_(eff) is a highly relevant parameter for colloidscience, biology and biotechnology. So far it only could be inferredfrom electrophoresis, restricted to particles smaller than the Debyelength (a≦3λ_(DH))³⁶. Unfortunately, many colloids are outside thisregime. As shown before, a similar size restriction does not hold forthermodiffusion. In many cases, the hydration entropy S_(hyd)contributes less than 15% (FIG. 8) and can be neglected at moderate saltlevels. Thus we can invert equation (2) to obtain the effective chargeQ_(eff) for spherical molecules from

$\begin{matrix}{Q_{eff} = {\frac{2T^{2}}{3\eta\; D}\sqrt{\frac{{ɛɛ}_{0}k^{3}S_{T}}{\beta\;\pi\;\lambda_{DH}}}}} & (3)\end{matrix}$

The effective charge derived from thermodiffusion measurements ofpolystyrene beads and DNA is plotted in FIG. 11 over several orders ofmagnitude in size. The effective charge of beads scales linearly withparticle surface with a slope confirming the effective surface chargedensity of σ_(eff)=4500 e/μm² which was inferred from electrophoresisonly for small particles. Average deviations from linear scaling arebelow 8% (FIG. 11 a). The effective charge inferred from thermodiffusionmeasurements of DNA using equation (3) scales linearly with DNA lengthwith an effective charge of 0.12 e per base pair. The length scaling isconfirmed over four orders of magnitude with an average error of 12%(FIG. 11 b). Thus thermodiffusion can be used to infer the effectivecharge with low errors for a wide range of particle sizes. This is evenmore interesting for biomolecule characterization since measurements ofthermodiffusion can be performed all-optically in picoliter volumes.

Conclusion.

We describe thermodiffusion, the molecule drift along temperaturegradients, in liquids with a general, microscopic theory. Applied toaqueous solutions, this theory predicts thermodiffusion of DNA andpolystyrene beads with an average accuracy of 20%. We experimentallyvalidate major parameter dependencies of the theory: linearity againstscreening length λ_(DH) and molecule hydrodynamic area A, quadraticdependence on effective charge and linearity against temperature.Measurements of thermodiffusion can be miniaturized to micron scale withthe used all-optical fluorescence technique and permits microscopictemperature differences to manipulate molecules based on their surfaceproperties (FIG. 7). The theoretical description allows to extract thesolvation entropy and the effective charge of molecules and particlesover a wide size range.

Infrared Temperature Control.

The temperature gradients used to induce thermodiffusive motions werecreated by aqueous absorption of an infrared laser at 1480 nm wavelengthand 25 mW power (Furukawa). Water strongly absorbs at this wavelengthwith an attenuation length of κ=320 μm. The laser beam was moderatelyfocussed with a lens of 8 mm focal distance. Typically, the temperaturein the solution was raised by 1-2 K in the beam center with a 1/e²diameter of 25 μm, measured with the temperature dependent fluorescencesignal of the dye BCECF¹³. Thin chamber heights of 10 μm to 20 μm andmoderate focussing removed possible artifacts from optical trapping,thermal lensing and thermal convection¹³. For temperature dependentmeasurements both the objective and the microfluidic chip were temperedwith a thermal bath. Imaging was provided from an AxioTech Variofluorescence microscope (Zeiss), illuminated with a high power LED(Luxeon) and recorded with the CCD Camera SensiCam QE (PCO).

Molecules.

Highly monodisperse and protein-free DNA of 50 bp, 100 bp, 1000 bp, 4000bp, 10000 bp and 48502 bp (Fast Ruler, fragments and λ-DNA, Fermentas)were diluted to 50 μM base pair concentration, i.e. the moleculeconcentration was between 1 μM (50 bp) and 1 nM (48502 bp). DNA wasfluorescently labeled by the intercalating TOTO-1 fluorescent dye(Molecular Probes, Oregon) with a low dye/base-pair ratio of 1/50.Carboxyl modified polystyrene beads of diameter 2 μm, 1 μm, 0.5 μm, 0.2μm, 0.1 μm, 0.04 μm and 0.02 μm (F-8888, F-8823, F-8827, F-8888, F-8795,F-8823, F-8827, Molecular Probes) were dialyzed (Eluta Tube mini,Fermentas) in aq. dest. and diluted in 1 mM Tris pH 7.6 toconcentrations between 3.3 aM (2 μm) and 2 nM (0.02 μm).

Concentration Imaging Over Time.

Either the method of concentration imaging¹³ or single particle trackingwere used to measure thermodiffusion at low concentrations, namely below0.03 g/l for DNA and 10⁻⁵ g/l for beads. At higher concentrations, wefound profound changes of thermodiffusion coefficients. DNA andpolystyrene beads smaller than 0.5 μm diameter concentration were imagedover time¹³ by bright field fluorescence with a 40× oil immersionobjective. Concentrations inferred after correcting for bleaching,inhomogeneous illumination and temperature dependent fluorescence¹³ werefitted with a finite element theory. The model captures all details ofboth thermodiffusive depletion and backdiffusion to measure D_(T) and Dindependently (see supplementary material). Measurements were performedin microfluidic chips 10 μm in height with PDMS on both sides¹³.

Single Particle Tracking.

Polystyrene particles larger than 0.5 μm in diameter were measured bysingle particle tracking due to the slow equilibration time and riskthat steady state depletion is disturbed by thermal convection. Thethermodiffusive drift was imaged with a 32× air objective at 4 Hz at aninitial stage of depletion in a 20 μm thick chamber. Averaging over thez-position of the particles removed effects from thermal convection. Thedrift velocity versus temperature gradient of 400 tracks were linearlyfitted by v=−D_(T)∇T to infer D_(T). The diffusion coefficients D of theparticles were evaluated based on their squared displacement, matchingwithin 10% the Einstein relationship.

The present invention is not limited by what has been particularly shownand described hereinabove. Rather the scope of the present inventionincludes both combinations and sub-combinations of the featuresdescribed hereinabove as well as modifications and variations thereofwhich would occur to a person skilled in the art upon reading theforegoing description and which are not in the prior art.

Infrared Heating.

The temperature gradients used to induce thermophoretic motions arecreated by aqueous absorption of a fiber coupled infrared solid statelaser (Furukawa FOL1405-RTV-317), with a wavelength of 1480 nm and amaximum power of 320 mW typically used at 25 mW. Water strongly absorbsat this wavelength with an attenuation length of κ=320 μm. The infraredlight is coupled out of the fiber to form a parallel beam with an 1/e²diameter of 1 mm. The beam position in the x/y plane can be adjusted bytwo galvanoometrically controlled infrared mirrors (Cambridge Technology6200-XY Scanner with Driver 67120). The laser beam is focussed frombelow the object stage by an infrared corrected aspheric lens with 8 mmfocal distance (Thorlabs, C240TM-C). Typically, temperature wasincreased by only 2 K in the heated focus.

Temperature Measurement.

The temperature gradient was measured via the temperature dependentfluorescence signal of the dye BCECF, diluted to 50 μM in 10 mM TRISbuffer. Details of bleaching correction and temperature extraction weredescribed previously¹³. From the total temperature dependence of BCECFof −2.8%/K, only −1.3%/K stems from pH drift of the used TRIS buffer.The remaining −1.5%/K are the result of thermodiffusion of the dyeitself, measured to be S_(T)=0.015/K with the concentration over timemethod described below.

“DNA” Image.

Measurement of the “DNA” profile in FIG. 7 was performed in a 60 μmthick chamber between glass slides, imaged with a 10× objective andheated to 2 K along the letters “DNA” with laser scanning. The chamberwas filled with a 50 nM solution of 1000 bp DNA stained with theintercalating fluorescent dye TOTO-1 (Molecular Probes) To switch fromdepletion to accumulation, the experiment was performed at roomtemperature or with the chamber cooled to 3° C., respectively.

Fluorescence Approaches.

Historically, methods used to measure thermodiffusion in liquids arebased on changes in refractive index upon change in soluteconcentration²⁷. Inherently, this signal is small for low soluteconcentrations near the limit of non-interacting molecules, even forintricate detection methods like thermal lensing or holographicinterference³⁸. Although operating at much smaller volume, the usedfluorescent microfluidic approach¹³ allows concentrations of 0.03 g/lfor DNA and reaches 10⁻⁵ g/l for single particle tracking. This isnecessary, since for example in thermodiffusion of DNA, we see profoundchanges at higher concentrations.

Thermodiffusion from Concentration Over Time.

Both DNA and polystyrene beads smaller than 0.5 μm in diameter weremeasured by imaging molecule concentration over time by bright fieldfluorescence. A more basic steady state method was describedpreviously¹³. Here, we refined it with a numerics theory to inferdiffusion coefficient D and Soret coefficient S_(T) independently.

Highly monodisperse and protein-free DNA of 50 bp, 100 bp, 1000 bp, 4000bp, 10000 bp and 48502 bp (Fast Ruler, fragments and λ-DNA, Fermentas)were used for the length dependent measurements. The DNA wasfluorescently labeled by the intercalating TOTO-1 fluorescent dye(Molecular Probes, Oregon) which shows 1000× fluorescence increase whenbound to DNA. The dye/base-pair ratio was low (1/50) to avoid structuralor charge artifacts from the bound dye. The fluorescence was observedwith an 40× oil objective. DNA stock solutions were diluted to 50 μMbase pair concentration, which corresponds to molecule concentrationsbetween 1 μM (50 bp) and 1 nM (48502 bp), respectively. Polystyrenebeads of diameter 0.2 μm, 0.1 μm, 0.04 μm and 0.02 μm (Molecular Probes,Oregon, F-8888, F-8795, F-8823, F-8827) were dialyzed (Fermentas, ElutaTube mini) in aq. dest., and diluted in 1 mM Tris pH 7.6 toconcentrations of 10 pM, 15 pM, 250 pM and 2 nM, respectively. DNAthermodiffusion measurements were performed in microfluidic chips 10 μmin height with PDMS on both sides¹³. They allow the measurement of smallvolumes in thin defined geometries. Polystyrene beads were sandwichedbetween a 1.25 mm thick polystyrene slide (Petri Dish, Roth) on thebottom and a plastic slide (170 μm thick, 1 cm×1 cm, Ibidi, Munich) onthe top and sealed with nail polish. For temperature dependentmeasurements, both 40× oil objective and microfluidic chip were temperedfrom below with a thermal bath. Note that temperatures well below 0° C.can be achieved as the microfluidic geometry reduces the probability ofwater to freeze.

Concentration of DNA was inferred from fluorescence images, that weremeasured with a 40× oil objective (NA=1.3) on an AxioTech Variofluorescence microscope (Zeiss), illuminated with a high power LED(Luxeon) and imaged with the CCD Camera SensiCam QE (PCO). The timeseries allows to correct for inhomogenous illumination and bleaching¹³.A time series dependent photobleaching means that an individualbleaching factor is determined for every image. This correction is ofadvantage for high precision measurements for protein thermophoresis. Ifsingle molecules were visible in fluorescence, time averaging was usedto average over particle positions.

Radial profiles were taken over time and combined to a space-time imageof both the thermophoretic depletion and back diffusion (FIG. 12 a,c).Fluorescence was adjusted for the temperature dependence of the TOTO-1dye determined independently with a fluorescence spectrometer as−0.5%/K. Typically, temperature in the solution was raised by 1-2 K inthe beam center with a 1/e² diameter of 25 μm.

The Soret coefficient S_(T) can be obtained from the steady stateprofile. Given the temperature at radius r obtained from temperaturedependent fluorescence, the concentration c(r) can be fitted with thesteady state thermophoretic profile¹³ given byc(r)=c ₀ e ^(−S) ^(T) ^((T(r)−T) ⁰ ⁾  (4)

with chamber temperature T₀ and bulk concentration c₀.

We can also obtain D and D_(T) independently by analyzing the build upand flattening of concentration profile over time after turning theinfrared laser beam on or off, respectively. Theory was provided fromfinite element model in radial coordinates (FEMLab, Comsol) over timewith boundary conditions of concentration obtained from the experiment.Comparison with experiment of the time course of thermophoreticdepletion reveals D_(T) (FIG. 12 c,d) and from the time course afterswitching off the heating source the diffusion coefficient D is obtained(FIG. 12 a,b). Results for diffusion coefficients obtained for DNAmolecules are shown in FIG. 13. Scaling of D for DNA larger than 1000 bpagree well with literature values and theoretical expectations³³.However for DNA molecules in the order of the persistence length (about150 bp) the power law exponent of −0.6 does not precisely fit themeasured values and a different scaling with an exponent of −1 isnecessary. A good description of DNA diffusion coefficients in the sizerange analyzed throughout this work is achieved with an intermediateexponent of −0.75.

Screening Length.

Debye-Hückel length was titrated by adding c_(s)=0 mM, 2 mM and 20 mM ofKCl to c_(T)=1 mM of TRIS buffer at pH 7.6 and calculated from

$\begin{matrix}{\lambda_{DH} = \sqrt{\frac{{ɛɛ}_{0}{kT}}{2{{\mathbb{e}}^{2}\left( {c_{S} + c_{T}} \right)}}}} & (5)\end{matrix}$

Changes in the effective charge of the molecules can be excluded atthese monovalent salt concentrations. For largest values λ_(DH)=13.6 nm,solely 0.5 mM Tris-HCl pH 7.6 buffer was used.

Thermodiffusion Using Single Particle Tracking.

For fluorescent polystyrene particles of large size (2 μm, 1 μm, 0.5 μm,Molecular probes, Oregon, F-8888, F-8823 and F-8827) a different methodhad to be used due to the increasing visibility of single particles,slow equilibration time and the risk that steady state depletion isdisturbed by thermal convection. Beads were dialyzed (Fermentas, ElutaTube mini) in aq. dest., and diluted in 1 mM Tris pH 7.6 toconcentrations of 3.3 aM, 25 aM and 0.2 pM, respectively.Thermodiffusion was measured in 20 μm thin chambers. A 1.25 mm thickpolystyrene slide (Petri Dish, Roth, Karlsruhe) was chosen as for thebottom of the chamber, while a plastic slide (170 μm thick, 1 cm×1 cm,Ibidi, Munich) was taken as cover slip. The low thermal conductivityensures constant temperature across the chamber. Chamber walls were madehydrophilic in a plasma cleaner (Harrick) for 10 min at 10 W electricalpower. As a result, adsorption of polystyrene particles to the plasticis low even at high salt concentrations. Addition of 2 μl bead solutionbetween the plastic sheets, followed by sealing the chamber with fastdrying nail polish leads to reproducible chamber heights of 20 μm.

Imaging was provided with an AxioTech Vario fluorescence microscope(Zeiss), illuminated with a high power LED (Luxeon) and imaged with theCCD Camera SensiCamQE (PCO). The center of the plain of view was heated8 K above room temperature with maximal temperature gradient of 0.2 K/μmand 1/e² spot radius of 50 μm Temperature was imaged with BCECF asdescribed before in a separate chamber. In FIG. 14 a finite elementsimulation of the experimental situation is shown using the commercialavailable Femlab software (Comsol). A 20 μm chamber is heated to 8 K inthe center. Due to low heat conductivity of the PS walls the temperatureprofile is homogeneous throughout the chamber height (FIG. 14 a). Due tothe thin chamber the convection speed is suppressed to negligible speedsof 5 nm/s at maximum (FIG. 14 b). The thermophoretic movement of theparticles was imaged with a 32× air objective and recorded at 4 Hz.Particles all over the 20 μm chamber could be equally tracked with acustom-written LabView program. Artefacts from toroidal thermalconvection are averaged out to a high degree as convective attractionnear the lower chamber wall cancelled with opposite convective repulsionnear the top of the chamber. Typically the velocity of 400 tracks wasplotted against radius and fitted with the drift velocity expected fromthermodiffusion according to thermophoretic drift v=−D_(T)∇T to find thethermophoretic mobility D_(T). Thermal fluctuations of the tracks wereevaluated based on their squared displacement to obtain the diffusioncoefficient D of the particles, which matched within 10% the Einsteinrelationship D=kT/(6πηa). This is expected since even for the worstcase, the chamber is 20-fold thicker than the diameter of the measuredparticles.

Electrophoresis.

The effective surface charge density was measured for 40 nm diameterbeads by electrophoretic drift in 400 μm thin and 5 cm long chambers(Ibidi, Germany). The velocity profile throughout the chamber height at5 V was taken from single particle tracking of 2 μm beads. At 80 μmheight the electroosmotic flow in a tightly sealed chamber is zero³⁹. Asexpected the velocity of particles in this plane saturates for particleslarger than 100 nm and is not related to effective charge³⁶. A highnumerical aperture oil objective has been used to analyze the velocityof 40 nm particles at the chamber surface at the same conditions. Theconstant velocity difference between chamber surface and plane of zeroelectroosmotic flow measured before has been used to calculate thepurely electrophoretic velocity.

ADDITIONAL REFERENCES REFERRED TO HEREIN ABOVE IN EXAMPLE 1

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Example 2 Determination of Hydrodynamic Radius and Interaction BetweenProteins

The thermo-optical characterization method of the present inventionallows also to quantify the hydrodynamic radius of proteins and evenmore important of complexes of biomolecules which are not connectedcovalently to each other. Thermophoresis provides a comparably robustand precise way to measure the hydrodynamic radius of molecules fromless than a nanometer up to a few microns. In comparison to the otherthermo-optical characterization methods the precision of this method isnot too sensitive on the geometry of measurement (e.g. height of theliquid layer) as it is the case for molecular interactions.

Data Acquisition:

A typical measurement can be described as follows:

Step 1:

A solution of fluorescently labeled molecules is brought into amicrofluidic measurement chamber (e.g. capillary, microfluidic chip).Fluorescence is excited and recorded with spatial resolution for lessthan 5 seconds on a CCD device with a frame rate between 40 Hz down to0.2 Hz (i.e. for fast diffusing molecules, a high frame rate is chosen).These image(s) provides the necessary information about the fluorescencelevel at 100% concentration. Then fluorescence excitation is turned off.

Step 2:

The Infrared laser heating is turned on. The immediately establishedlocal spatial temperature distribution causes the molecule drift tolower or higher temperatures, depending on the particular molecule to beanalyzed. The laser is focused in a way that temperature gradientsbetween 0.0 and 5 K/μm are achieved. The temperature gradient has beencalibrated once and it is not necessary to repeat this calibration everytime an experiment is performed. The maximal temperature elevation isbelow the temperature which is known to cause damage to the molecules ordisintegrate their interaction. Depending on the thermophoreticproperties of the molecules in the solution (i.e. if they move fast in athermal gradient or slow) the infrared laser heats the solution for 5seconds up to 100 seconds. After this period of time the infrared laseris turned off

Step 3:

After the spatial temperature distribution has vanished (typically 2-50ms) the fluorescence excitation is turned on and fluorescence isrecorded with the same frame rate used in the first step of fluorescenceimaging. This time the redistribution of the molecules is imaged for 5seconds up to 50 seconds. The exact time depends on the velocity withwhich the molecules diffuse (i.e. the time it takes them to equalize 90%of the concentration gradient established by thermophoresis).

Data Processing—Photobleaching:

The fluorescence images have to be corrected for photobleaching. Sincethere is no spatial temperature profile in the solution whilefluorescence images are taken, the bleaching correction is possible withhigh precision (i.e. high precision is possible since the rate ofphotobleaching is temperature dependent).

Therefore, the fluorescence at a edge of the measurement chamber (i.e. aspot as far away from the heated center as possible), werethermophoresis during step 3 was negligible (i.e. for a person skilledin the art, this is where the temperature gradient during laser heatingwas lower than 0.001 K/μm), is evaluated to determine the photobleachingfrom the image series taken in step 3. If photobleaching is present, thefluorescence will decrease from image to image. The individual factorfor each image is used to correct all images for bleaching. Anotherpossibility is to calculate the bleaching for every single pixel fromthe images taken in Step 1. The bleaching rate per pixel can be used tocorrect every pixel from step 3 images for the photobleaching effect.

Data Processing—Inhomogeneous Illumination Correction and Normalizationto 100% Concentration:

All images taken in step 4 are divided by a single or all images takenin step 2 and multiplied by 100. This way a correction for inhomogeneousillumination is achieved and the fluorescence is normalized to 100%concentration.

Data Processing—Determining the Hydrodynamic Radius:

From the first images of the step 4 image series the concentrationdistribution is extracted. A software tool evaluates the Diffusioncoefficient (or multiple Diffusion coefficients in case of a mixture)which describes the experimentally measured relaxation of theconcentration gradient. Using the Stokes-Einstein relation thehydrodynamic radius is inferred from the diffusion coefficient.

In Particular, the Above Described Experiment was Conducted for a Sampleof GFP as Follows:

The thermo-optical properties of two samples of Green Fluorescentprotein (GFP) are measured with the devices of the present invention. 2μl of GFP (5 μM, 1×PBS buffer) is pipetted on an object slide. Thesample is sandwiched by pitting an cover slip (round 12 mm diameter) ontop. The liquid spreads uniformly in between the glass surfaces an thechamber is sealed off by using nail polish. This prevents the liquidfrom rapid evaporation, which would in turn lead to a comparably strongflow of liquid in the chamber. This sample is placed on a device shownin FIG. 1 a. The measurement steps and data processing steps areperformed as described above. The same experiment is performed with asecond sample containing 5 μM GFP and 10 μM of a GFP binding antibodyfragment, specifically binding to GFP. In both cases, first thefluorescence is recorded without laser heating. Then the fluorescenceexcitation is turned off and the IR-laser radiation is turned on (themaximum temperature is kept below 35° C. (i.e. about 15° C. aboveambient temperature (about 20° C.) to avoid denaturation or damage tothe protein). The laser is turned off after a few seconds of heating andthe fluorescence excitation is turned on at the same time. Therelaxation of the spatial fluorescence distribution (i.e. concentrationdistribution) to a homogeneous state is recorded for a few seconds. Ascan be observed from FIG. 31, in the sample with the two interactingspecies (i.e. GFP and the antibody fragment) the fluorescence profileneeds more time to relax. This is explained by slower diffusion of thelarger complex. The time evolution of the fluorescence profile isanalyzed via a software tool (selfmade Software, Labview, NationalInstruments) to determine the diffusion constant. By using theStokes-Einstein relation, a hydrodynamic radius is attributed to thediffusion constant. In case of the free GFP this is 5 nm and the complexhas an radius of 10 nm.

Example 3 Detection of Interactions Between Biomolecules andDiscrimination of Nucleic Acids by Size

The thermo-optical characterization of the present invention providesthe means for fast all optical biomolecule analysis. Present methods fordetection and quantification of biomolecular interactions are very timeconsuming which means that the time needed for an analysis is on theorder of 30 minutes up to hours. The present invention can detect andquantify biomolecular interactions within 1 second up to 50 seconds. Theterm interaction comprises interaction between biomolecules (e.g.protein, DNA, RNA, hyaluronic acids etc) but also between modifiednanoparticles/micro beads and biomolecules. A typical experiment todetect/quantify interactions can be described as follows:

Step 1a, Background Measurement:

The sample buffer without fluorescently labelled samplemolecules/particles is filled in the microfluidic chamber and thefluorescence is measured, while the excitation light source is turnedon.

Step 1b, Determination of Fluorescence Level Before Laser Heating:

An aqueous solution of a fluorescently labelled sample (e.g.biomolecules, nanoparticles, microbeads whereas all of them have aspecific affinity for other biomolecules) at a given concentration isfilled in a microfluidic chamber (preferably a capillary) whichpreferably guarantees a defined height of the chamber. Fluorescence isexcited and recorded with (CCD-Camera) or without (Photomultiplier tube,Avalanche Photodiode) spatial resolution for less than 10 seconds on aCCD device or photomultiplier with exposure times of 25 milliseconds upto 0.5 seconds. Then the fluorescence excitation is turned off.

Step 2, Starting of Infrared Laser Heating:

In the following the infrared heating laser is turned on and the spatialtemperature distribution is established within a few milliseconds withinthe solution. The temperature gradient has been calibrated once and itis not necessary to repeat this calibration every time an experiment isperformed. In particular a setup were infrared heating and fluorescenceimaging are performed through the same optical element from one side isadvantageous for the stability of the optical and infrared foci.

It is of advantage that in the experiment the decrease of fluorescencedue to photobleaching is lower than 5%.

The maximal temperature elevation is below the temperature which isknown to cause damage to the molecules in the solution or disturb theirinteraction (e.g. temperatures between 1 and 5° C. above ambienttemperature).

Depending on the thermophoretic properties of the molecules in thesolution (i.e. if they move fast in a thermal gradient or slow) theinfrared laser heats the solution for 5 seconds up to 100 seconds.

Step 3, Recording of the Spatial Fluorescence (i.e. Concentration)Profile:

After this period of time the fluorescence excitation is turned on andimages are recorded with the same frame rate and length as described instep 1b. Step 3 is the last acquisition step necessary for evaluation ofthermo-optical properties.

For detection and quantification of interactions more measurementsfollowing the protocol described previously are necessary. Step 1a isrepeated with sample buffer and in step 1b the aqueous solution of afluorescently labelled sample is mixed with an amount of the biomoleculewith which the interaction should be detected or quantified. For thedetection of an interaction it is necessary to mix the fluorescentlylabelled sample with a sufficient amount of binding partner so that asubstantial amount of the fluorescently labelled molecule is in thecomplex with the binding partner. If the strength of the interactionshould be quantified in terms of a dissociation or association constant(Ka, Kd), than the procedure described previously has to be conductedwith varying concentrations of binding partner (e.g. 0.1×-10× theconcentration of the fluorescently labelled binding partner). This meansthat a titration of binding partner should be performed.

Processing the Raw Data:

For a linear bleaching correction it is necessary to wait for theback-diffusion of all molecules following the end of step 3. Thisincreases the time consumption of the analysis dramatically. For preciseand fast measurements it is of advantage to determine the strength ofbleaching from image to image and correct every individual image withits own bleaching factor. For a precise bleaching correction it isimportant that the temperature gradient at distance from the heat spotis low (e.g. below 0.001 K/μm). The images taken in step 1b are used tocorrect all images for inhomogeneous illumination. In case fluorescenceis recorded without spatial resolution (e.g. avalanche photodiode orphotomultiplier) photobleaching is corrected best by determining oncethe bleaching characteristic of a certain dye without heating laser in acontrol experiment.

Data Evaluation:

Qualitative detection of interaction: From the image series the spatialfluorescence distribution of the reference experiment (i.e.fluorescently labelled molecule/particle without binding partner) andthe second experiment (i.e. were the binding partner is present) isextracted. The fluorescence is plotted versus the distance from the heatspot. An averaging is only possible for pixels with the same temperatureand same distance. The spatial concentration distribution is obtained bycorrecting the fluorescence intensities for the respective temperaturedependence of the dye. With knowledge of temperature dependence of thefluorescent dye and the spatial temperature distribution, the effect ofa decreasing fluorescence due to temperature increase can be corrected.For the qualitative detection of interaction as well as theirquantification a correction for temperature dependency is not necessary,and the spatial fluorescence distribution is sufficient. This enables usto use any fluorescent dye on the market without characterization of itstemperature dependency.

The values of the fluorescence profile are integrated up to the distancewere the temperature is below 10% of the maximum temperature (e.g. 70μm). The integrated values are compared and a change give a preciseindication if there is an affinity between the substances at theconcentrations used, since the interaction changes the thermo-opticproperties (e.g. thermophoretic mobility, surface size and chemicalgroups on surface). In most cases the interaction leads to higherfluorescence (concentration) at higher temperatures.

In case the whole cross-section of a capillary is heated (i.e. usingcylindrical lenses to give the IR laser beam a ellipsoidal shape, whichheats a cross section of a capillary homogeneously), the intensity of alot more pixels from the centred heat spot can be averaged since allpixels at same distance to the heated line have the same temperature.This is advantageous for high precision measurements. In casefluorescence is recorded without spatial resolution the fluorescencechange in the centre of the heat spot/line is measured, whereas it isagain advantageous to heat the whole cross section. In general if morethan a single frame is recorded in step 1b and 3 an integration ofmultiple frames is possible.

For a quantification of affinities the same procedure is performed forall experiments at various concentrations of non fluorescent bindingpartner. The result of the integration for the reference experiment(i.e. without binding partner) is subtracted from the integrated valuesobtained for the different concentrations of binding partners. From thisevaluation on gets the amount of interacting complexes in arbitraryunits. By dividing the values by the value were binding is saturated therelative amount of complexes at a certain concentration of binder isobtained. From these dataset also the concentration of free nonfluorescent binding partner can be determined and the strength of theinteraction can be quantified in terms of association or dissociationconstant (see FIG. 25).

Example 3a Interaction of Proteins

FIG. 25 shows a quantification of interaction between biomolecules. 100nM of a fluorescently labelled antibody in 1×PBS buffer(anti-Interleukin 4, Sigma-Aldrich) are titrated with various amountsinterleukin 4 1×PBS buffer (0-300 nM IL4 Sigma-Aldrich). (left).Approximately 200 nl of the sample mix are soaked into a capillary of 40nm inner diameter (World Precision Instruments). The capillary issituated on a device as shown in FIG. 27. Fluid drift is prevented byclosing the valves on both sides of the capillary. The measurement isperformed with the device shown in FIG. 20. The spatial fluorescencedistribution in steady state is measured using the previously describedprotocol. Three results for 5 nM, 80 nM and 300 nM are shownexemplarily. After each measurement the capillary is flushed withapproximately 5 μl of 1×PBS buffer. The binding of IL 4 to the antibodychanges the signal dramatically from fluorescence decrease to afluorescence increase. Integration of the fluorescence profile up to 80μm (distance from the heated centre, see procedure described in thisexample herein above) allows to determine the number of complexes insolution. (right) The concentration of free Interleukin 4 can becalculated plotted versus the concentration formed complex. These datacan be fitted to determine the K_(D).

Example 3b Discrimination of DNA by Size and Interaction of DNA Strands

Qualitative detection of interaction, using a modified protocolaccording to this example 3, is shown in FIG. 5. Here the concentrationprofiles of 20 base pair DNA has been compared to 50 base pair DNA at amaximum temperature increase of 10° C. (at ambient temperature of 20°C.). In a second experiment 20 bases single stranded DNA is comparedwith 20 base pair double stranded DNA. All four experiments wereperformed in 1×SSC buffer the following way: 2 μl of sample are pipettedon an object slide (Roth, 1 mm thick) an sandwiched between an coverslip of 12 mm diameter. The aqueous solution spreads uniformly inbetween the two glass surfaces yielding a thin sheet of water with aheight of approximately 20 μm. The liquid sheet is sealed by using nailpolish. This avoids rapid evaporation of the sample. The microfluidicchamber is placed on the object stage of the thermo-optical setup (e.g.FIG. 1 a) and imaged via a 40× oil objective (NA 1.3, Zeiss). The laserfocus is positioned so that it is approximately in the center of thefield of view and has a half width of about 20 μm. Only a single pixelof a CCD camera used for detection of the fluorescence. This pixelmeasures the fluorescence in the center of the heat spot. Thefluorescence is recorded for approx. 1 seconds without laser heating,then the IR Laser is turned on while fluorescence is still recorded.After 20 seconds of laser heating the measurement stopped. As can beseen from FIG. 5 single stranded DNA can be discriminated from doublestranded DNA and DNA of different length can be discriminated within atime span of a few seconds. Bleach correction is not performed in thismeasurement strong change in concentration is observed.

Example 4 Detection of Binding of PEG Molecules to Nanoparticles

As mentioned previously it is also possible to detect the binding ofmolecules to larger inorganic particles or nanocrystals using theprocedure described previously. Inorganic CdSe particles (core diameterabout 12 nm) have been modified with a varying numbers (1 up to 3) ofPoly-ethylen-glycol (PEG) of different molecular weight. 2 μl of sampleare pipetted on an object slide (Roth, 1 mm thick) and sandwichedbetween a cover slip of 12 mm diameter. The aqueous solution spreadsuniformly in between the two glass surfaces yielding a thin sheet ofwater with a height of approximately 20 μm. The liquid sheet is sealedby using nail polish. This avoids rapid evaporation of the sample. Themicrofluidic chamber is placed on the object stage of the thermo-opticalsetup (e.g. FIG. 1 a) and imaged via a 40× oil objective (NA 1.3,Zeiss). The laser focus is positioned so that it is approximately in thecenter of the field of view and has a half width of about 100 μm. Themaximum temperature increase was determined to 5° C. above ambienttemperature. The spatial fluorescence profile is recorded as describedpreviously for the detection of biomolecular interactions. Also the rawdata are processed as described previously. To measure the number orsize of PEG molecules bound to the nanocrystals it is sufficient tocompare the spatial fluorescence profiles obtained with the protocoldescribed previously. However, a correction for the temperaturedependent decrease of the fluorescence allows a quantification in termsof the Soret coefficient. FIG. 26 shows that the Soret coefficientincreases linearly with the number of PEG molecules bound to thenanocrystals. The slope of the increase depends on the molecular weightof the PEG. FIG. 26 shows that the binding of single molecules of thesize of a protein is detectable.

Example 5 Thermophoresis of Proteins

An example how the conformation, structure and surface of a moleculeeffect the thermo-optical characteristic of said molecules and how thesecharacteristics may be measured, detected or characterized is given inthe following:

A sample of fluorescently labelled BSA (Bovine Serum Albumin, Fermentas)is transferred in a microfluidic chamber (e.g. capillary). Thetemperature of the whole sample volume is adjusted by a Peltier elementin thermal contact to the solution (the microfluidic chamber is placedon a device shown in FIG. 27). The Peltier element is used to regulatethe “ambient temperature” of the solution. It does not create a spatialtemperature distribution. The adjustment of temperature is importantbecause the thermo-optic properties (e.g. surface properties,conformation) at varying ambient temperatures (i.e. proteinconformations) should be measured. The thermo-optic properties aremeasured following the protocol described previously for biomoleculeinteractions (step 1 to step 3, without addition of binding partnerssince only intramolecular properties are measured). Also the processingof the raw data follows the procedure described for the detection ofmolecule interactions. The thermo-optical properties are evaluated bydetermining the concentration profile in steady state. The thermo-opticproperties are plotted as Soret coefficient ST as shown in FIG. 28. TheSoret Coefficient is obtained by correlating the concentration profilein an exponential fashion (c=c₀e^(−S) ^(T) ^((T−T) ⁰ ⁾) to thetemperature distribution. The Soret-Coefficient is sensitive to changesin the interplay between the amino acids of the protein and the watermolecules. At low temperatures the molecules are accumulated in a regionof elevated temperature, which corresponds to a negative Soretcoefficient. At increasing temperatures the accumulation of themolecules changes (i.e. the Soret coefficient increases). This can bereadily explained by changes in the conformation of the molecule (e.g.hydrophobic groups or loops rearrange). As can be seen from FIG. 28 thesign of thermophoresis changes from negative values at lowertemperatures to positive values (i.e. depletion) at higher temperatures.The sudden jump to positive Soret coefficients correlates very well withthe temperature were thermal denaturation occurs (i.e. 50° C.). In therange of body temperature (i.e. 30° C.-40° C.) the thermo-optical signaldoes not significantly change. An explanation for this unexpectedbehaviour is that the protein is evolutionary designed to be functionalwithin this temperature range. Since there is a tight structure-functionrelationship in nature, the structure is preserved in this temperaturerange. The values shown in FIG. 28 are corrected for the temperaturedependence of the fluorescence dye.

The local temperature increase in the system causes a change influorescence which is not purely caused by changes in concentration dueto thermophoresis. Since the temperature of transition from negative topositive values is important for measurements of absolute proteinstability, a correction for the temperature dependence of fluorescenceis advantageous. In application were differences in stability should bedetected (e.g. small molecule binding to a protein) the correction fortemperature dependence of the fluorescence is not necessary. Theargument that protein structure/conformation is measured is supported byFIG. 28 b where the experiment is started at high temperatures. Even attemperatures below the thermal denaturation temperature the Soretcoefficient is positive. This is based on the slow refolding timecompared to the speed of measurement, which is faster than 50 seconds.After a certain time span the thermo-optical ST values shown in FIG. 28a are also obtained again in the measurement shown in FIG. 28 b. As acontrol the temperature of the system is increased again and thenegative ST values are obtained as expected at temperatures of about 40°C.

Example 6 Detection of Conformational Changes, Like Denaturation ofProteins

-   a) An example where the denatured form of a protein is distinguished    from the native form without any correction for the temperature    dependence of the fluorescence dye, given in FIG. 15. An aliquot of    a fluorescently labelled Bovine Serum Albumin is heated up to 90° C.    for 10 minutes, which is well above the temperature of denaturation    and the protein cannot refold. Also the thermo-optic characteristics    of a native aliquot (i.e. not heated to 90° C.) is measured.    Starting with the native sample, approximately 200 nl of the sample    are soaked into a capillary of 40 nm inner diameter (World Precision    Instruments). The capillary is situated on a device as shown in    FIG. 27. Fluid drift is prevented by closing the valves on both    sides of the capillary. The measurement is performed with the device    shown in FIG. 20. The spatial fluorescence distribution in steady    state is measured using the previously described protocol. After    each sample the capillary is flushed with approximately 5 μl of    1×PBS buffer. The experiment is performed as described for the    measurement of biomolecular interactions (Step 1-Step 4). The    experiment is performed twice with different infrared laser powers    (i.e. maximal temperatures of 5° C. and 10° C. above ambient    temperature are employed). Afterwards the sample with the denatured    protein is measured. Again three experiments with different laser    powers are used. In FIG. 29 the fluorescence is plotted as a    function of the distance to the heat source (i.e. laser focus) is    shown for the native and denatured form at two different laser    powers (i.e. maximum temperatures of 5° C. and 10° C.). In both    cases there are two contributions to the fluorescence change. First    there is an increase or decrease in concentration (for the native or    denatured form, respectively) and secondly there is a decrease in    fluorescence due to the temperature dependence of the fluorescence    dye. For a qualitative comparison (i.e. to distinguish between a    native and denatured form.) a correction for the temperature    dependence of the dye is not necessary. In all cases shown in FIG.    29 the fluorescence decreases. But as expected the decrease in    fluorescence for the native protein is not as strong decrease    observed for the denatured form. This is readily explained by the    negative Soret coefficient of the native protein at 20° C. ambient    temperature (see also FIG. 28), which leads to an accumulation of    molecules at higher temperatures. This counteracts the decrease in    fluorescence caused by the temperature dependence of the    fluorescence. At higher laser powers (i.e. a maximum temperature    increase of 10° C.) the difference between native and denatured form    is even stronger because the thermophoretic accumulation and    thermophoretic depletion, for the respective form, get stronger.    Also smaller conformational changes can be detected on the basis of    different strength of thermophoresis. A different direction of    thermophoretic movement is advantageous but not necessary.-   b) A sample of fluorescently labelled bovine serum albumin (BSA) has    been split in two parts. One is only exposed to ambient    temperatures, while the other half is heated up to 100° C. for    several minutes (i.e. irreversibly denatured). The thermo-optical    properties of both samples (native and denatured) are measured at    800 mA power of the infrared laser (i.e. maximal temperature    increase of 20° C.). As can be seen from FIG. 30, the fluorescence    of the denatured protein is lower than the fluorescence of the    native protein. This is explained as follows. The fluorescence dye    of both samples shows the same decrease in fluorescence due to the    increase in temperature (i.e. temperature sensitivity of the    fluorescence). But the denatured protein shows a positive    thermophoretic mobility (i.e. moves to the cold), while the native    protein has a negative thermophoretic mobility (i.e. moves to the    hot). The accumulation at elevated temperatures is the reason, why    the decrease in fluorescence is lower for the native protein, while    the denatured protein is, in addition to temperature dependency,    depleted from the region of elevated temperature. Interestingly, by    approaching the denaturing temperature (i.e. 50° C.) of the protein    the amplitudes of the native and denatured protein approach each    other an are essentially the same. This means that by measuring the    amplitude of the fluorescence change an comparison to the reference    sample allows to detect the melting temperature of a protein and to    discriminate between native and denatured form of a protein. And to    detect a shift in melting temperature due to interactions of the    protein with other biomolecules or small molecules (e.g. drug    candidates).

Example 7 Optothermal Trap/Thermooptical Trap

In the following, silica particles are employed as illustrativeparticles/beads to be thermo-optically trapped by the method of thepresent invention. It is understood that the described method can alsobe employed for the thermo-optical trapping of other molecules, likebiomolecules or lipid vesicles (as also illustrated in a furtherexample).

Silica particles (1 μm diameter, plain, Kisker Biotech) are diluted1/100 in distilled water. 2 μl are pipetted on an object slide (Roth, 1mm thick) an sandwiched between an cover slip of 12 mm diameter. In thefollowing the term bead is used as a synonyme for particle. The aqueousbead-containing solution spreads uniformly in between the two glasssurfaces, yielding a thin sheet of water with a height of approximately20 μm. The liquid sheet is sealed by using nail polish. This avoidsrapid evaporation of the sample. The microfluidic chamber is placed onthe object stage of the thermo-optical setup (as e.g. illustrated in theappended FIG. 1 a) and imaged via a 40× oil objective (NA 1.3, Zeiss).The laser focus is positioned so that it is approximately in the centerof the field of view and has a half width of about 100 μm. Then the IRLaser is turned on and heats the solution to 10° C. above ambienttemperature (20° C.) at the maximum of the spatial temperaturedistribution in the center of the IR laser focus. The images seriesshown in FIG. 33 illustrates the process of particleaccumulation/trapping. In the beginning (first image of FIG. 33, top ofthe page), without laser heating, the beads are almost equallydistributed. The black circle shows the position of the laser focus. Thefollowing images show the development of the particle distribution inthe next three seconds after the heating laser is turned on. Theparticles show a directed movement to the region of elevated temperatureat the laser focus, which can be also referred to as negative Soreteffect or negative thermophoresis. Surprisingly, silica particle shownegative thermophoresis at room temperature. The particles are trappedin the center of the temperature distribution at the laser focus. Theparticle experiences a potential well created by the spatial temperaturedistribution. The directed motion to the region of highest temperatureis explained by the particle's tendency to minimize its energy ofsolvation. The position of the particle is not exactly in the center ofthe heat spot, since the thermal fluctuations push the particle out ofits position. Appended FIG. 34 illustrates that the beads are trappedwithin the region of highest temperature even when the stage (i.e.sample) is moved with speeds of millimeters/second relative to the fixedlaser focus. By using the thermo-optical trap, particle can be movedarbitrarily and can also be concentrated. After confining antibodymodified beads to the heat spot, an interaction between the particlesdue to a binding of a single antigen in the solution to more than onebead can be detected.

Another approach of a thermo-optical characterization in accordance withthis invention is shown in FIG. 32. Silica particles (1 μm diameter,plain, Kisker Biotech) are diluted 1/1000 in distilled water. Thedilution factor is empirical. The dilution is such that only a singleparticle is observed in a region of approximately 400 μm times 400 μm. 2μl are pipetted on an object slide (Roth, 1 mm thick) an sandwichedbetween an cover slip of 12 mm diameter. The aqueous bead-containingsolution spreads uniformly in between the two glass surfaces yielding athin sheet of water with a height of approximately 20 μm. The liquidsheet is sealed by using nail polish. This avoids rapid evaporation ofthe sample. The microfluidic chamber is placed on the object stage ofthe thermo-optical setup (e.g. FIG. 1 a). The laser focus is positionedso that it is approximately in the center of the field of view and has ahalf width of about 100 μm. Then the IR Laser is turned on and heats thesolution to 10° C. above ambient temperature (20° C.) at the maximum inthe center of the IR laser focus. Due to the high dilution, a singleparticle is trapped in a potential well created by a spatial temperaturedistribution (s. FIG. 32 a). As silica particles show a negativethermophoresis the well is deepest at high temperatures (i.e theparticles minimize their energy of solvation at high temperatures). Thesingle silica particle fluctuates in the potential well since thethermal fluctuations push the particle out of its position. Thefluctuations are recorded via a CCD camera (at t=1 s, 2 s, 3 s, 4 s, 5s, 6 s, 7 s) and the positions are tracked by Software (selfmadeSoftware, Labview National Instruments, detecting the pixel with highestintensity) with nanometer resolution (see FIG. 32 b). A histogram iscalculated from the positional information (see FIG. 32 c). The width ofthe distribution is very sensitive to the thermo-optical properties ofthe particle. If molecules bind to the surface of the particle, theeffective potential for the bead changes and the amplitude of thefluctuations increases or decreases. By observing the amplitude changeover time, a kinetic binding curve can be measured. In a microfluidicsystem such an experiment is performed as follows: a solution containinga 1/1000 dilution (distilled water) is flushed or soaked into acapillary (see FIG. 27). The valves at the end of the capillary areclosed. A single particle (modified (e.g. coated) with an antibodyspecific to a certain antigen, e.g. Interleukin 4) is trapped and thefluctuations of the particle are detected for 10 seconds to 100 seconds.The beads is surrounded by pure buffer solution. In a next step thebuffer is exchanged with a buffer containing the respective antigen.While the buffer is exchanged the bead is still trapped. After exchangeof buffer the fluctuation of the same bead as before are recorded. Thechange in fluctuation amplitude is used to detect interactions betweenantibody and antigen. Its change over time is used to measure bindingkinetics.

By using a device as illustrated in the appended FIG. 24, a temperaturegradient can be generated in solution, by scanning lines (e.g. 10)perpendicular to each other in the solution. Where these lines cross, atemperature maximum is observed. Points on the scanned line have anintermediate temperature, while spaces in between the lines representtemperature minima (e.g. ambient temperature if spaces in between thelines are sufficiently wide). The silica particle described above willmove to equilibrium positions on the crossing points of the heatedlines. By moving the temperature grating all particles will movesimultaneously. In addition the fluctuation of all beads may be measuredsimultaneously.

Example 8 DNA Melting Curves

Standard protocol for the measurement of melting curves (using thedevice shown in e.g. FIG. 1 a, 1 b, 16 to 18, 20 to 24 or 37):

-   -   Raman-Laser 1455 nm; coupled to galvanometric mirrors via fibre    -   No collimator after fibre, Laser-beam hits mirrors divergently;        mirrors are reflecting the laser-beam onto a lens; laser beam is        focused to the chamber via the lens.    -   Laser on/off is controlled via moving the laser in an out of the        field of view via the mirrors.    -   Preparations:        -   Coverslips (170 μm) thick, one with a diameter of 12 mm the            other quadratic 24×24 mm rinsed with deionized water, rinsed            with ethanol, then again rinsed with deionized water.        -   Dilution of solutions:            -   10 μM Tamra in 1×SSC            -   Hairpin from 100 μM in MilliQ-water diluted to 10 μM, 1                μM, 100 nM, or less in SSC-Buffer (1×, 0.5×, 0.1× or                less).            -   add the detergent TWEEN20 to an end volume of 0.01%                (only if there is unspecific adsorption).    -   Adjustment:        -   Check if everything is ok with the microscope (apertures,            filters)        -   10 μM Tamra (tetramethylrhodamine) in 1×SSC (150 mM NaCl, 15            mM Na3-citrate, pH 8.1), with 0.01% TWEEN 20; volume of 2 μl            into chamber built with 2 Coverslips (170 nm thick), sealed            with nail polish        -   Wait until nail polish is dry        -   Add immersion oil onto the upper coverslip        -   Put chamber onto the measurement stage        -   Focussing fluorescence image by nearly closing the aperture        -   Find laser spot        -   Focus laser        -   Defocus laser by increasing the distance between lens and            chamber        -   Adjust laser focus in a way that a broad and appropriate            temperature distribution is established        -   Move laser spot with galvanometric mirrors out of the field            of view in such a way that the influence of the laser to the            fluorescence image is minimal→save this settings to the            measurement program (avoid to cross the zero point on the            voltage scale of the galvanometric mirrors)        -   Measure the room temperature    -   Measurement:    -   Use the following steps for the measurement of temperature as        well as for the measurement of melting curves:    -   Conduct the measurement with the trigger-programme.    -   Settings: 40× oil immersion-objective, 8×8 Binning, 10 ms        exposure time (→28 Hz readout rate)    -   To do manually:    -   1. Laser on    -   2. Light source on (open shutter in case of HXP), write down the        settings of the light source    -   3. Start the trigger program    -   4. Laser off    -   Light source on

The measurement is based on the detection of fluorescence and thereforemay be conducted in a device according to appended FIGS. 16-18 and/or20-24. As the exact timing of measurements is preferable, the useddevices like CCD, IR-Laser and light source are synchronised by the useof an electronic trigger signal. As the used CCD-camera (Andor Luca) hasa trigger output port, the IR-Laser control element and the light sourcecontrol element are synchronised with the trigger signal of theCDD-camera. In particular the second high level of the CCD triggeroutput signal is taken as the zero point of time of the measurement. Inthe following the exposure time of the CCD-camera is 10 ms and theminimal time span between two images is determined by the frame rate ofthe CCD-camera. As chamber a 2 μl solution of the fluorescently labelledsample molecules (e.g. 10 μM tetramethylrhodamine (TAMRA) in 75 mM NaCl,7.5 mM Na3-citrate, 0.01% TWEEN 20, pH 8.1) is sandwiched between two170 μm thick glass coverslips with diameter of 12 mm and sealed withnail polish. This results in a thickness of the chamber of about 20 μm.Then the chamber is moved to the measurement device and the optics arefocussed to the chamber.

Brief Description of the Sequence of the Measurement:

Before the measurement starts, the fluorescence background of themeasurement device is recorded in “Step 0”. As this background ischaracteristic for the used device and may not change during a long timeperiod this step is preferably done only once to characterize the useddevice.

-   Time t=0: Step 1: a first fluorescence image of the spatial    fluorescence distribution in the chamber is recorded.-   Time t=20 ms Step 2: the IR-Laser is switched on-   Time t=60 ms Step 3: a second fluorescence image of the spatial    fluorescence distribution in the chamber is recorded.

After these steps the measurement is finished and the raw dataprocessing and the data evaluation is conducted.

Detailed Description of the Sequence of the Measurement:

Step 0, Background Measurement:

A sample buffer without fluorescently labelled samplemolecules/particles is filled into a microfluidic chamber and thespatial fluorescence distribution in the chamber is measured with theCCD-camera, while the excitation light source is turned on.

Step 1, Determination of Fluorescence Level at Ambient TemperatureBefore Laser Heating:

An aqueous solution of a temperature sensitive dye (e.g. TAMRA) at agiven concentration (e.g. 10 μM) is filled in the chamber whichpreferably provides a defined height of the chamber. Fluorescence isexcited with the light source (LED) and recorded with spatial resolutionwith the CCD-camera at ambient temperature.

First the CCD-camera is started and the first high level of the cameratrigger output signal is used to synchronise the IR-Laser controlelement and LED control element with the CCD-camera. For thesynchronising a measurement card from e.g. National Instruments can beused.

For good illumination the LED is turned on with the use of the cameratrigger signal a short time before the CCD-camera records a firstfluorescence image I₀(x,y). Therefore during the exposure time of theCCD-camera of 10 ms the fluorescence excitation light source has reachedits steady state level of light output. When the camera starts itsrecording the output trigger signal of the camera is the second time atthe high level state. This second high level state of the output signaldetermines the zero point of time t=0.

Step 2, Starting of Infrared Laser Heating:

The infrared heating laser is turned on at time t=20 ms and the spatialtemperature distribution is established within a few milliseconds withinthe solution. The temperature distribution has been calibrated once in away that for example all temperatures between 30° C. and 90° C. arepresent in the recorded image and it is not necessary to repeat thiscalibration every time an experiment is performed.

Step 3, Recording of the Spatial Fluorescence Profile with InfraredLaser Heating:

At t=60 ms, 40 ms after IR-Laser irradiation was started, a secondfluorescence image I₁(x,y) is recorded with the CCD-camera with anexposure time of 10 ms.

After the CCD-camera has taken the second picture, the first and thesecond picture are saved to the hard disk of a PC for processing the rawdata and for data evaluation. Then the measurement process is finished.

Processing the Raw Data and Data Evaluation:

Because of the short time of measurement no correction for bleaching maybe necessary. The images I₀ and I₁ are corrected against camerabackground. Then calculating the ratio K(x,y)=I₁(x,y)/I₀(x,y) for eachpixel of the fluorescence images (second image divided by the firstimage, both background corrected) ensures the removal of artefacts froman inhomogeneous illumination. As the temperature dependence F(T) (FIG.15) of the dye TAMRA is known from a calibration experiment in afluorimeter, the spatial temperature distribution T(x,y) (FIG. 3 c) canbe derived from to the ratio K(x,y).

As the timing of measurement is the same for the temperature measurementand for the measurement of the melting curve, both measurements can beconducted at one time in one chamber if the emission spectrum of the dyefor the temperature measurement (e.g. TAMRA) can be well separated fromthe emission spectrum of the fluorescent label of for example themolecular beacon (e.g. HEX as fluorophore (see appended FIG. 6) andDabcyl as a quencher).

Example 9 Detection of Covalent and Non-Covalent Modifications ofNanoparticles

An example for the detection of covalent and non-covalent modificationis shown in FIG. 35. Nanoparticles (i.e. nanocrystals or quantum dots)have been obtained from Invitrogen. Particles were purchased which havecharged polymer coating to be stable in aqueous solution (diameter 12nm). In addition particles with covalently coupled strepavidin werepurchased (diameter 21 nm), as well as a biotinylated 40 bases singlestranded DNA. The following samples have been prepared: First unmodifiednanoparticles diluted to 1 μM concentration in 1×SSC (Saline-SodiumCitrate) buffer. Secondly, streptavidin coated nanoparticles diluted in1×SSC buffer to 1 μM concentration. Thirdly, streptavidin coatednanoparticles diluted in 1×SSC buffer to 2 μM concentration were mixedwith 2 μM 40 bases biotinylated single stranded DNA (IBA GmbH,Gottingen) in 1×SSC buffer in a 1/1 ratio. All three experiments wereprepared in 1×SSC buffer in accordance with the following protocol: 2 μlof sample are pipetted on an object slide (Roth, 1 mm thick) ansandwiched between an cover slip of 12 mm diameter. The aqueous solutionspreads uniformly in between the two glass surfaces leading to a thinsheet of water with a height of approximately 20 μm. The liquid sheet issealed with nail polish to avoid rapid evaporation of the sample. Themicrofluidic chamber is placed on the object stage of the thermo-opticalsetup (e.g. FIG. 1 a) and imaged via a 40× oil objective (NA 1.3,Zeiss). The laser focus is positioned so that it is approximately in thecenter of the field of view and has a half width of about 20 μm. Theexperiments were conducted as described herein above for the detectionof interactions, with a maximum temperature increase of 5° C. above roomtemperature. A correction for temperature dependent fluorescence hasbeen performed for precise measurement of the Soret Coefficient from thespatial concentration distribution. (i.e. the temperature dependency ofthe fluorescence of the nanocrystal has been determined in anindependent fluorimeter experiment). As can be seen from FIG. 35, theSoret coefficient is positive and larger for the streptavidin coatednanoparticle than for the nanoparticle without protein. Furthermore thebinding of a single single-stranded DNA molecule to the particle isdetected as a change in Soret coefficient. This is interesting, sincethe short flexible DNA molecule does not contribute substantially to thesize of the nanoparticle (as the streptavidin does). Sincethermophoresis is sensible to changes of the surface properties uponbinding of the DNA molecule, the binding of the comparatively small DNAmolecule to the particle can be detected.

Example 10 Thermophoresis and Thermophoretic Trapping of Lipid Vesicles

Texas-Red DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt), a fluorescently labelled phospholipid, is usedfor staining the vesicles. The lipid is added to the process of vesicleformation with approximately 1 mol percent with respect to the mainconstituting phospholipids. The following stock solutions were used forpreparation of the vesicles by electro swelling:

Lipid stock solution:

-   -   2.5 mg/ml DPhPC (1,2 Diphytanoyl-sn-Glycero-3-Phosphocholine) in        Chloroform (CHCl₃)    -   1-2% stearylamine        -   1% fluorescent lipid            (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,            triethylammonium salt (Mol. Probes)

Sucrose stock solution:

-   -   300 mM Sucrose in distilled water

Buffer solution:

-   -   150 mM KCl, 20 mM MES, pH 5

The final vesicle solution is diluted 1/100 in distilled water. 5 μl ofthe dilution were pipetted on an object slide. The droplet wassandwiched in between the object slide and a round 12 mm diametercoverslip and placed on the measurement apparatus. Fluorescence wasobserved through an oil objective using the setup from 1a, andalternatively the setup from FIG. 19. In the latter case, IR-Laserheating is performed through the same objective. The maximum temperatureincrease was about 15° C. The temperature profile had a half width of 20μm. Images before and after infrared laser heating were taken. Tworepresentative images before and 10 seconds after infrared laser heatingare shown in FIG. 39. The vesicles are attracted by heat spot due tonegative thermophoresis. They are accumulating in the centre of the heatspot. The catchment area is dependent on the width of the temperatureprofile (i.e. a sufficient strong temperature gradient is necessary todrive directed particle motion toward the heated centre within a finitetime). The trapping of vesicles has several, important applications,e.g. vesicles (as well as cells) can be transported and moved within asolution. Also, the fluctuation of single vesicles in the potential well(i.e. created by temperature increase) can be observed. Since theamplitude of fluctuations is dependent on the properties of the vesicle,any changes, like protein binding to vesicles or the activity of amembrane protein, e.g. an ion-pumping membrane protein can be detectedas a change in fluctuation amplitude. The sign of the thermophoreticmotion (e.g. attracted to the heat centre or repelled by the heatedcentre) is dependent on the properties (e.g. charge, size, surfacemodification, protein binding). When a vesicle which is normallyattracted by the region of highest temperature is repelled from theheated centre, this is indicative for a change in the properties of thisvesicle. This behaviour may be observed after changing the buffer aroundthe vesicle to a solution containing, e.g. a binding partner. Also thebehaviour of two samples containing vesicles in buffer and vesicles inbuffer with binding partner (or membrane protein activating substance,e.g. ATP) may be compared or observed. Finally, the differences in thethermophoretic properties can be used to sort vesicles or cells.

The invention claimed is:
 1. A method for measuring inter- and/orintra-molecular interactions of particles in a solution comprising: a)providing a sample with marked particles in a solution; b) excitingfluorescently the marked particles and detecting a first fluorescence ofthe excited particles; c) irradiating a laser light beam into thesolution to obtain a spatial temperature distribution in the solutionaround the irradiated laser light beam; d) detecting a secondfluorescence of the particles in the solution at a predetermined timeafter irradiation of the laser into the solution has been started, ande) characterizing the inter- and/or intra-molecular interactions of theparticles based on the first and second detections, wherein thebrightness of said fluorescence is detected with a photomultiplier tube,a photodiode or a single pixel with a CCD in the centre of the laserbeam.
 2. The method of claim 1, wherein both fluorescence detection andinfrared laser focussing are performed through a single optical unit. 3.The method of claim 2, wherein both fluorescence detection and infraredlaser focussing are performed through a single objective.
 4. The methodof claim 1, wherein the predetermined time is within the range of 1 msto 250 ms.
 5. The method of claim 1, wherein the detection time is inthe range of 1 ms to 50 ms.
 6. The method according to claim 1, whereinthe spatial temperature distribution in the solution around theirradiated laser light beam is 0.0 to 5 K/μm.
 7. The method of claim 6,wherein the spatial temperature distribution in the solution around theirradiated laser is 0.0 to 2 K/μm.
 8. The method of claim 1, wherein thelaser beam is irradiated through an optical element into the solution.9. The method of claim 8, wherein the optical element is a single lens.10. The method of claim 1, wherein the solution further comprisesmeasuring the temperature distribution in the solution around theirradiated beam by detecting the color intensity of a temperaturesensitive dye.
 11. The method of claim 10, wherein the temperaturedistribution is determined based on detected fluorescence of thetemperature sensitive dye, wherein the solution comprising saidtemperature sensitive dye is heated by the irradiated laser beam and thefluorescence spatial fluorescence intensity is measured substantiallyperpendicular around the laser beam.
 12. The method of claim 11, whereinthe spatial temperature distribution is 0.001 to 10K/μm.
 13. The methodof claim 11, wherein said fluorescence is detected with a CCD camera.14. The method of claim 1, wherein the predetermined time is within therange of 0.5 s to 250 s.
 15. The method of claim 1, further comprisingdetecting a distribution of fluorescence, wherein a change in thedistribution of fluorescence within the spatial temperature distributionin the solution during the predetermined time measures a change inconcentration of the particles.
 16. The method of claim 1, wherein theparticles are selected from (i) a biomolecule; (ii) a nanoparticle;(iii) a microbead; and (iv) combinations thereof.
 17. The method ofclaim 16, wherein the biomolecules are selected from the groupconsisting of proteins, peptides, nucleic acids, protein-nucleic acidfusion molecules, PNAs, and locked DNAs (LNAs).
 18. The method of claim1, wherein the laser light has a wavelength of 1200 nm to 2000 nm. 19.The method of claim 1, wherein the laser is a high power laser of 0.1 Wto 10 W.
 20. The method of claim 19, wherein the laser is 4 W to 6 W.21. The method of claim 1, wherein the solution is an aqueous solutionwith a particle concentration of 1 atto Molar to 1 M.
 22. The method ofclaim 21, wherein the solution is an aqueous solution with a particleconcentration of 1 atto Molar to 100 μM.
 23. The method of claim 1,wherein the solution is a saline solution with a concentration in therange of from 0 to 1 M.
 24. The method of claim 1, wherein the spatialtemperature distribution is between 0.1° C. and 100° C.
 25. The methodof claim 24, wherein the temperature gradient is created within 0.1 μmto 500 μm in diameter around the laser beam.
 26. The method of claim 1,wherein the solution is provided with a thickness in direction of thelaser light beam from 1 μm to 500 μm.
 27. The method of claim 1, whereinthe detection of the fluorescence is detected within a range of from 1nm to 500 μm in the direction of the laser beam.
 28. The method of claim1 wherein the particles to be measured are selected from the groupconsisting of biomolecules, nanoparticles, microbeads, organicsubstances, inorganic substance and/or combinations of these.
 29. Adevice for measuring inter- and/or intra-molecular interactions ofparticles in a solution according to claim 1, wherein the devicecomprises: a receiving means for receiving marked particles within asolution; means for fluorescently exciting the marked particles; meansfor detecting the excited fluorescence in said solution wherein selectedfrom a photomultiplier tube, a photodiode or a single pixel with a CCDin the centre of the laser beam; and a laser for irradiating a laserlight beam into the solution to obtain a spatial temperaturedistribution in the solution around the irradiated laser light beam. 30.The device of claim 29, wherein the laser and the means for detectingthe excited fluorescence are arranged such that fluorescence imaging andinfrared laser focussing are performed through a single optical unit(1).
 31. The device of claim 30, wherein the fluorescence imaging andinfrared laser focussing are performed through a single objective. 32.The device of claim 29, wherein the means for fluorescently exciting themarked particles is an LED.
 33. The device of claim 29 wherein the laseris a high power laser of 0.1 W to 10 W.
 34. The device of claim 33,wherein the laser is 4 W to 6 W.
 35. The device of claim 29, wherein thedevice further comprises an optic for magnifying the detected region.36. The device of claim 29, wherein the device further comprises anoptic for focusing or defocusing the laser beam.
 37. The device of claim36, wherein the optic is a single lens.
 38. The device of claim 29,wherein the detecting means (31) is a CCD camera.
 39. The device ofclaim 29, wherein the detecting means (31) is a photo diode.